• Understand what is reasonable for you to do. You need to know your limits before you decide what you can and cannot do.  These limits may have to do with your skills or your time, but sometimes they are based on your role in the lab.  As a grad student or post-doc, you shouldn’t be doing the work of a tech; as a tech, you shouldn’t be expected to produce data like a post-doc.  Be aware of your role and job description, and be ready to articulate your objections if you think a particular assignment is beyond the scope of your responsibilities.

• Make your decision based on data, not emotion. You need to be organized enough in your work and your scheduling that you know how much you are doing and when.  That way, when you beg off an engagement by saying that you are too busy, you can explain exactly what you are busy doing.  If you’re feeling frazzled when a request comes in, take some time to think about it, ideally after you’ve had some time to relax: what sounds impossible right now may not look so tough tomorrow.

• Try making it sound like a “maybe”. Supervisors are often taken aback by a direct “no” from an employee.  Even if you know that a certain task is not possible for you to complete, try to start a conversation about it instead of flatly refusing.  Explain why you are concerned about completing the task: is it the time frame?  the skills required?  a problem with equipment availability?  Your supervisor may be able to solve some of these problems for you; or, there’s a good chance they will agree with your assessment and stop pressuring you to complete the task.

• Be gracious in defeat. No matter how tactful you are, there is a chance you will be overruled or otherwise “forced” into doing something you’d rather not.  This is not the time to mope; this is the time to complete the task or assignment as quickly and efficiently as possible, so you can get back to your other work.  Feel free to drop a few hints to your supervisor about how it is as complicated as you expected, or how you feel rushed or pressured, so they don’t think you were refusing for no reason; but be sparing, since they will most likely remember the complaint, instead of your hard work.

Have you found a good way to say no when the tasks start piling up?  Tell us about it in the comments!

Background
Before I read the WIRED article and the linked NIST Technical Note, I have to admit that I was blissfully unaware of how laser pointers worked. In simple terms, laser pointers (other than most red ones) utilize an infrared laser diode to produce infrared light, which is then manipulated by crystals to adjust its wavelength to the desired visible wavelengths, such as green, blue, violet, and orange-red (read more here). The problem is that the conversion of the original infrared light into visible light isn’t 100% efficient, and the shoddier the craftsmanship of the laser, the less efficient it likely is, and the less likely the device contains an IR filter to remove the leftover IR wavelengths. In the NIST Technical Note, the authors found that their cheap green laser pointer was outputting 20 mW of total power, with over 90% of that power still in the IR spectrum.  To put this in prospective, with this level of power output, protective eyewear must be worn while the laser is in use in order to be in compliance with our laser safety protocols and OSHA.

The dangers of IR
So why be concerned by the IR leakage? The NIST authors state it best:

“Intense infrared radiation poses a grave danger to humans and animals because, being invisible, it does not activate the blink reflex. Thus, a considerable dose of infrared radiation can enter the eye and cause retinal damage, imperceptible to the victim before serious damage occurs… ”

In addition to a conference room setting, where both the visible and the IR wavelengths are reflected off the screen,

“… many windows in modern commercial and industrial buildings have infrared reflective coatings to reduce solar heating of the interior. If a green laser is directed on such a window, the green light will pass through it, but any infrared emitted by the GLP will be reflected. This is a substantial hazard. Any differential reflection of infrared vs. green light poses such hazards, since the green light that travels with the infrared light may not be sufficiently intense to activate the blink reflex.”

The NIST IR detector
In the NIST Technical Note, the authors create a low-tech apparatus using a CD as a diffraction grating to separate the visible and infrared wavelengths being emitted by the laser pointer. They then take pictures of the diffraction pattern with both an IR sensitive and IR filtered cameras and compare the two pictures to reveal the contaminating IR wavelengths. While I was attempting to replicate their approach with the random contents of my desk, my colleague (and the aforementioned friendly neighborhood physicist) Dr. Chris Dombrowski started questioning what I was up to.

Using an IR Thermometer as an IR detector
In the past, I’ve written about the utility of IR thermometers in the laboratory, and consequently I have one handy at my bench. Once I described what I was doing to Chris, he pointed out that I didn’t need to separate the various wavelengths of light (possibly) being emitted by my laser pointer, as long as I had a sensor that was only responsive to IR wavelengths, and that the IR thermometer should fit that bill. Sure enough, when I shined my cheap green laser off the detector of my IR thermometer, the thermometer went nuts, registering temperatures as high as 85°C, while no external components of the laser pointer were warm to the touch.

We headed off to the bat cave (fluorescent microscope room) to use some more sophisticated equipment to verify what we found. Similar to the NIST results, my cheap green laser pointer was outputting approximately 15 mW of total power, with 10 mW in the IR spectrum. A survey of the lab turned up a second, more expensive green laser pointer that was outputting approximately 0.5 mW of IR radiation. (We believe this laser pointer has better alignment of the crystals then the cheap laser pointer, but still lacks a protective IR filter in the design.) When we tested the effect of the second laser pointer on my IR thermometer, we found a shift of only a couple of degrees when the laser was on. In addition, Chris used optical filters to show that filtering out the green light of my cheap laser pointer still resulted in a large response from the IR thermometer, while using an IR filter eliminated the thermometer’s response to the laser pointer. Taken all together, we feel pretty confident that my commercial IR thermometer is unaffected by the visible wavelengths of light (as it should be), while the high levels of contaminating IR energy being emitted by the cheap laser pointer are detected by the artifactually high temperature readings of the thermometer.

When using the IR thermometer as an IR laser detector, ensure that nobody’s eyesight is at risk, that you are actually aiming at the IR sensor of the thermometer (the bottom opening), and to take several (~10) readings with the laser pointer “on” to reduce the possibility that the potential IR beam simply missed the detector. In this photo, dry ice "fog" was used to visualize the visible laser beam, and should not be used while testing a laser pointer.

Conclusions
The readings on an IR thermometer can tell you if your laser pointer is also emitting IR wavelengths, and in theory the magnitude of the reading should bear some correlation to the amount of IR being emitted from the laser pointer. However, because we don’t know many of the parameters of the IR detector in the thermometer, we think it would be premature to draw any direct correlation between the IR power outputs of the lasers we tested and the temperature readings of the thermometer. However, like the apparatus designed by the NIST scientists, the IR thermometer can serve as a qualitative measure of the contaminating IR energy emitted by the laser pointer. IR thermometers are an inexpensive piece of equipment that have tremendous utility in the research lab, but because they are also used as no-touch children’s thermometers and in the heating and cooling industry, with a little effort these devices are also available to a large number of people outside of a laboratory setting. (In other words, if you don’t want to buy one of these, your building’s maintenance personal are likely to have one that they might let you use for a few minutes.) In addition, evaluating a laser pointer using an IR thermometer is very simple with no elaborate set up required, but please do use common sense and be careful not to orient the devices in such a matter that you might shine the laser in your eyes.

Before you even get to grow your actual culture, you have to take a loop from your frozen stock, streak it and grow colonies then pick colonies and grow a starter culture. What a drag.

Sounds like time for a shortcut, so here it is…. make a cell bank. A cell bank is essentially a rack-full of small, uniform, frozen starter cultures for the culture you grow regularly.

Making a cell bank

To make a cell bank, just streak your culture, pick a single clone and grow up a flask of cells until just before they are saturated, then add glycerol to a final concentration of 10%. Next aliquot it into screw-top tubes before freezing at -80°C. Make the aliquot sizes 100^th of the culture size you normally grow, and judge how many aliquots you’ll need by how often you normally find yourself needing to grow the culture.

Now when you want to start a culture, you just need to take a tube from your cell bank, thaw slightly, then add your frozen starter culture straight into the waiting flask full of growth medium.

Not only does this save time in setting up the culture, but since each starter culture in the cell bank has exactly the same cell density, you can expect your culture growth to be reproducible; just monitor the growth the first time you grow a culture from the cell bank and you should get very similar growth curves for each subsequent culture. Also, since all of the cultures you grow from your cell bank will come from the same clone, this can make your results more reproducible.

Don’t forget to label

Now you can have a rack in the -80 containing row upon row of starter cultures for each strain you culture routinely. Just make sure to label them properly. It can be tedious labeling aliquot after aliquot, so my approach is to use a color-coding/symbol system. Give each batch its own, easily recognizable marking (e.g. a red, blue or green stripe – stripes, make it very easy to mark many tubes quickly -, or if you need more options, use circles, squares etc), then maintain an Excel file/paper sheet that lists each cell bank with its symbol, strain, date of production, cell density etc. And you’re ready to go.

Do you use cell banks already, or do you have another system for streamlining your routine culture preps?

1. Tools for Detecting the phosphorylation state of proteins

Over the course of a research project, eventually you’ll want to know what phosphorylation state a protein is in, whether it’s a result of in vivo regulation or in vitro modification. The four following techniques rely on protein electrophoresis. Once you’ve got your protein of interest in solution – whether in a crude mixture of proteins, or purified, you have the following options:

Stains to detect all phosphoproteins

This method is right for you if you want to keep yourself free and open to all potential phosphoproteins or phosphorylation states of a protein that might come your way; you can detect all phosphoproteins without restricting yourself to a single protein, or a single motif or residue. The Pro-Q Diamond Phosphoprotein Gel Stain from Invitrogen will do just that, and can be visualized with a laser scanner or UV transillumination. You can even go a step further and instead use the Blot Stain kit to detect phosphoproteins on a PVDF or nitrocellulose membrane. Then wash away the stain and multiplex with western blotting.

Pros: You get to see any and all phosphoproteins, on a gel or a membrane. Fast and easy procedure.
What to watch for: Only sensitive to the low nanogram range- better than Coomassie but sometimes not good enough for precious little protein. Also, detecting all phosphoproteins may be biting off more than you can chew if you electrophorese and stain complex and concentrated mixtures of phosphoproteins- you might get more of a “smear” than clean bands of the protein you’re looking for.

Antibodies for specific amino acids

Say you’re interested in proteins phosphorylated specifically on serine residues, not threonine, tyrosine, or anywhere else. You could use an antibody that would only bind to such phosphoserine proteins. I’ve used the PhosphoDetect Phosphoserine Detection Kit from Calbiochem, complete with four clones of antibodies to detect phosphorylated serines, as well the surrounding amino acid environment. That point is very important to keep in mind- the antibody will not bind to just any phosphoserine present in a protein, but must recognize and have affinity for adjacent “epitopes” as well.

Pros: High specificity and high sensitivity, as expected with western blotting.

What to watch for: “Epitope” requirement can be bothersome. I wanted to use casein, a protein rich in phosphoserines as a positive control/standard. Casein is highly phosphorylated at sites surrounded by acidic amino acids, and I learned that these antibodies unfortunately detect phosphoserines in environments with basic amino acids. I ultimately couldn’t get the antibodies to detect a single speck for casein! Also, with regards to this method and the next two- antibodies aren’t cheap!

Antibodies for specific proteins and specific phosphorylation sites

The first two methods had the potential to detect a multitude of phosphoproteins- what if you were interested in just one? You could use antibodies targeted to that single protein, and moreover, specific phosphorylation sites within that protein. The Phospho-PKC Antibody Sampler Kit from Cell Signaling Technology helped me when I was working on protein kinase C (PKC), to differentiate between the different isozymes and where they were phosphorylated (Ser744, Thr505, Ser916 to name a few).

Pros: Specificity for one single protein and phosphorylation site, and the sensitivity of western blots all rolled into one.

What to watch for: Limiting. For this method (and the next), the more specific and narrow you get with your detection methods… what happens if what you think you’re looking for, turns out not to be what you’re looking for?

Antibodies to detect specific targets of phosphorylation

Let’s take the previous method and flip it around- say you weren’t interested in PKC per se, but you were instead more concerned with what PKC phosphorylated and which of its targets you could find. You’d be interested in detecting the specific motif in proteins to which PKC binds while phosphorylating the serine residue within it. Surely enough, there are antibodies for those as well, like the Phospho-(Ser) PKC Substrate Antibody from Cell Signaling Technology.

Pros: Once again, specificity and sensitivity, what’s not to love?

What to watch for: As before, in the context of this example- what happens if your protein of interest turns out to be possibly phosphorylated at a threonine instead of a serine, and this antibody doesn’t detect it? What happens if your protein of interest turns out to not be phosphorylated by PKC in certain conditions… or at all?

2. Tools for determining activity, affinity and specificity of protein kinases

Just as important as the proteins being phosphorylated are the protein kinases doing the phosphorylating. What are the kinetic parameters of a kinase in given conditions? Will it phosphorylate a novel protein of interest or won’t it? These are important questions you may come across over the course of your research.

Radioisotopes

Hot stuff! Take a protein kinase and give it either a protein or peptide substrate, use normal ATP spiked with a bit of radioactive gamma-32P-ATP, and watch the results light up. Anything phosphorylated will be detectable with high sensitivity to radiometric methods such as phosphor storage screens and laser scanners, and even greater sensitivity with liquid scintillation counting (LSC).

Pros: Very high sensitivity and broad detection- as long as a kinase phosphorylates a protein and transfers that terminal 32P-phosphate over, it’s detectable- regardless of which amino acid is phosphorylated, the nature of the surrounding epitope, antibody recognition, and so forth.

What to watch for: The joys of working with lucite shields, double-gloves, Geiger counters, radiation safety offices, and everything else that comes with an ionizing-radiation source that can potentially be pretty harmful!

Peptides and P81 paper

Sir Philip Cohen and his group at Dundee wrote a Nature Protocols paper referring to this method, quite correctly, as the “gold standard” of protein kinase quantification. Simply, it involves reacting a protein kinase with a peptide containing a motif known to be recognized by the kinase and, importantly, rich in basic amino acids and hot ATP. The reaction mixture is then spotted onto negatively-charged P81 phosphocellulose paper, allowing the positively-charge peptide to bind and everything else- including any autophosphorylated kinase and unreacted ATP- to wash away. The hot peptides on the paper can then be detected by phosphor storage screens or LSC.

Pros: Simple enough to get the hot ATP, and you can get the peptides virtually anywhere- potentially even at your own core facility.

What to watch for: Some peptides can be expensive depending on where they’re purchased, surprisingly even more expensive than their whole-protein counterparts. Using only those peptides rich in basic residues can also limit which kinases you can assay. Conceivably, you can use acidic peptides and DEAE (positively-charged) paper but I’ve never come across that. Further, what happens when you use neutral or somewhat hydrophobic peptides that won’t interact with ionic paper at all?

Proteins and precipitation, or electrophoresis

Limited by peptides? Take Sir Philip’s protocol above and substitute a short peptide for a whole protein. In fact, it’s actually a reaction more similar to physiological conditions. The problem, however, is that if you had issues getting certain peptides to stick to P81 paper, then a whole protein could be a nightmare. Instead, precipitate the protein chemically, such as with TCA, to filter paper, leaving the unreacted ATP to remain in solution and wash away. Or, better yet, load your reaction mixture into an acrylamide gel and electrophorese away. You’re ultimately looking for a radioactive protein band at the correct molecular weight in the gel, and can even Coomassie stain it (safely- keep in mind you’re working with a hot gel) to help you find what you’re looking for.

Pros: Greater choice of protein substrates to be phosphorylated. With electrophoresis, you’re sure to identify your target of interest, at the correct molecular weight, appearing as a radioactive band.

What to watch for: By using TCA washes or electrophoretic running buffer, and then staining and destaining a gel, you may be spreading radioisotopes into even more solutions that require careful disposal.

Non-radiometric assays

As the safety demands and distaste for using radioisotopes grow, many enterprising biotech companies are coming up with ways to eliminate that dependency. New kits are continually being developed that rely on fluorometric or colourimetric detection of protein kinase reactions. My grad lab was one of the first that tested the Omnia Kinase Assays by Invitrogen on non-recombinant and non-human kinases. The assays use a peptide with an unnatural amino acid fluorophore- upon phosphorylation of the peptide, magnesium is chelated between the phosphosite and the fluorophore, enhancing fluorescence. What was especially nice about this assay as opposed to others was that, instead of merely detecting the endpoint of phosphorylation, the Omnia Kinase assay principle allows phosphorylation progress to be tracked in real-time. I also tried out the IMAP FP kinase assay by Molecular Devices, which showed promise but ultimately didn’t suit my needs. That assay uses peptides which once again contain a fluorophore, but rely upon fluorescence polarization. Upon phosphorylation, the phosphopeptide binds to a trivalent metal (M3+) nanoparticle, and fluorescence polarization increases. Unfortunately, what I also discovered was that virtually anything with phosphates can bind to these metal nanoparticles, occupying binding sites and quenching the fluorescence polarization of the peptide: unreacted ATP, ADP, AMP, cofactors, phosphoproteins- all of which you can easily find when using crude lysate or using high concentrations of ATP.

Pros: Sensitive, convenient kits, eliminating the need to use radioisotopes.

What to watch for: “Dime a dozen” may refer to the abundance of non-radiometric kinase assays being developed by companies these days, but certainly not the price. These can easily cost orders of magnitude above what is required for a simple assay using hot ATP.

3. Tools for determining activity, affinity and specificity of protein phosphatases

So far, we’ve looked at proteins that have phosphates on them, and the kinases that put them on there. Just as important and certainly not to be forgotten, are the protein phosphatases which dephosphorylate them.

Go Green!

Ekman and Jager presented, back in 1993, a method now commonly used to assay subnanomolar inorganic phosphate hydrolyzed from phosphoproteins by phosphatases. The principle is based upon the strong green colourimetric complex formed between the malachite green dye, molybdate, and free orthophosphate. As with protein kinase assays, a protein or peptide substrate can be used for a protein phosphatase, but in this case, the substrate is phosphorylated to begin with, and presumably loses phosphate as the incubation continues. At the end of the desired incubation time, the reaction is quenched with the dye reagent, and absorbance at 600 nm is read in a simple spectrophotometer.

Pros: Sensitive (hey, it’s subnanomolar!), easy mix-and-read method. Conceivably one type of assay for any protein phosphatase with any phosphopeptide or phosphoprotein substrate.

What to watch for: If you thought peptides were expensive, phosphopeptides can be even more so! Try to find cheap, bulk proteins to use as substrates, like casein. Also, that dye can definitely make a staining mess- which can actually be less trivial and more serious than it sounds if you spill it in your spectrophotometer.

The yellow glow of para-nitrophenyl phosphate

Remember 2nd-year undergraduate biochemistry lab? If so, then you’ll recall the classic lab that involves, in one way or another, alkaline phosphatase reacting with a molecule to produce an increasing yellow colour over time. That molecule is actually para-nitrophenyl phosphate (pNPP), and its hydrolysis product, para-nitrophenol (pNP) is the yellow-coloured molecule which absorbs at 400 nm. You can use a simple spectrophotometer to detect hydrolysis in real-time.

Pros: Cheap, simple, real-time method.

What to watch for: Not the highest sensitivity. Moreover, pNPP is a rather small molecule and not a physiological peptide or protein with recognition motifs- not all phosphatases will bind and hydrolyze it, and so it may not be usable for all phosphatases.

Radiolabeled protein and peptide

Earlier, we saw how peptides and proteins can be radiolabeled by reacting them with a protein kinase and hot ATP. With a bit of ambition, skill, and luck, those peptides or proteins can be purified and used as substrates for phosphatases. The assay principle is the same, but in reverse- instead of detecting radioactivity being incorporated into a protein by a kinase, the aim is to detect radioactivity being removed from a protein by a phosphatase. In addition to making your own radiolabeled protein and peptide, you can also purchase commercially-available ones.

Pros: Sensitive, universal, all the advantages of assay protein kinases with radioisotopes now applied to protein phosphatases.

What to watch for: The use of radioisotopes and everything that goes with them, again.

I hope you found this useful, although it doesn’t even scratch the surface of available tools! Use this as a foundation and go see for yourself what best suits your needs! If you have anything to add, or any questions about the tools I have described, please be sure to leave a comment.

Next, we’ll take a closer look at protein kinases, beyond what we covered in their introduction in Part I.

Several years ago, candidates received offers if they met six or seven out of 10 of the qualifications. Now, without having nine out of 10, it remains difficult to get an offer. So the question is “what’s hot”?

Medical Devices has been going strong despite the weak job market of the past couple of years. R&D, Life Sciences and Biotech have been picking up in recent months. However, there is no sense of urgency in these departments to make a hire. Unless they find the perfect (10 out of 10) background, they are electing to stockpile their money and not invest in training or development of someone who does not meet every requirement.

How does this help you?

First, set your expectations with these facts in mind. If you do not meet all of the criteria for a position, you can still apply and make a case for yourself, but try not to get frustrated if you don’t receive a callback.

Second, if you do have the skills in the job description, take the time necessary to make sure every key word from the job description is peppered throughout your CV. The number of positions that fit your background perfectly are likely manageable enough to accomplish this. You get one shot to make a first impression – don’t risk it by asking the hiring company to read between the lines on your resume. Spell it out for them. (If you don’t, someone else will.)

Third, prep and plan for your interviews. Be on your game, and review the company’s web site and job description before the interview. Highlight skills and experiences that are relevant to the company.

Fourth, keep your chin up. The right job will come along and when it does the process will move quicker than you can imagine. When hiring managers like what they see on the resume (i.e. when it very closely reflects the job description), we have been seeing offers made and accepted within days of the initial resume submission. Hiring managers want to hire, but they are being more selective than ever. They have been asked to do more with less over the past two years and now that they have an opportunity to hire, they want to be sure they find the right person.

What are you seeing and hearing in your job search? Have you experienced these things? Has your experience been different? Please share your thoughts as this is one of those topics where input from you will be crucial to make this post mean something. Anything you can share about industries or skill sets that are desirable will help everyone else who reads this.

After what has potentially (likely?) been years of data collection and a month or two of writing, re-writing, wailing and gnashing of teeth, your first paper has been hammered into shape. Hopefully the process has yielded something metaphorically closer to Michelangelo’s David than Mr. Potato Head. Either way, it’s time to send your creation out into the word and see your hard work in print. Here is a brief description of the publication process

The cover letter
In the old days, manuscripts were submitted by mail, and one wrote a cover letter so that the person who opened the package didn’t have to figure out the contents on their own. Nowadays, manuscripts are submitted electronically, but cover letters are still part of the package in one form or another. For some journals it still looks like a letter, and for others the various elements are entered into fields on a webpage.

The purpose of the cover letter is three-fold: to explain to the editor the significance of the work (ie – why their journal should publish your work), to suggest other experts in your field to review the work, and to exclude other scientists that may have competing projects or other conflicting interests. (There are some other legalities that are taken care of here, but they aren’t all that interesting.) The latter two areas are strictly between you and the editors at the journal, but know that these are only suggestions – if the editor feels that you have “stacked the deck” by excluding all of the most qualified scientists, they have the right to send it out to those that you have requested not review the work. The significance of the work may or may not be confidential – some journals now send this statement along with the abstract when asking potential referees if they are willing to review the work. From what I have seen, however, the journal will explicitly state which sections are confidential and which will be shared with the referees.

The importance of the cover letter grows, generally speaking, as the impact factor of the journal rises. At the top of the heap, journals like Cell, Science, and Nature are only interested in publishing novel, ground-breaking research and they receive far more manuscripts than they can publish. The cover letter for these journals needs to convince the editors that your manuscript fits the bill, otherwise your manuscript may never even be read. Well, it will, but not by them…

The editors
Different journals are organized differently, but in general terms there are at least two levels of editors – ones at the top that have the final say on publications, and a second tier of editors that actually handle the review of the manuscripts, often called managing editors. At the top journals these positions are all filled by full-time PhDs that work for the journal, while most journals have a much larger staff of academic lab heads (PIs) who volunteer a portion of their time to the journal as editors.

Once you and your mentor submit your manuscript to a journal, somebody takes a look at the cover letter, abstract, and key words and tries to decide which of the managing editors has the expertise to manage the review of the manuscript. This editor doesn’t need to have the level of expertise that the referees’ posses, but he or she must be familiar enough with the field to know who to ask to be a referee, and to make educated decisions when there is a difference of opinion between two referees or between a referee and the authors.

Once the manuscript (with cover letter) has been sent to the managing editor and that editor has agreed to handle the paper (if the editor is a volunteer), then the first decision that he or she makes is if the manuscript is suitable for the journal. If this editor feels that your work isn’t consistent with the journal’s mission, then he or she can reject the paper without it even going out for review. This is called an editorial rejection, and there isn’t much you can do about it. It could be because:

• they don’t believe your topic is “sexy” enough for their journal,
• they don’t think it constitutes a significant enough advance in the field even if the topic is sexy,
• they think the discipline is wrong (trying to publish a biochemistry paper in The Journal of Cell Biology, for example),
• or they feel that the work is derivative (it has already been published before).

The only good thing about an editorial rejection is that it generally happens quickly. If the editor is undecided about a manuscript’s suitability for their journal, he or she may send out “feelers” to lab heads, which consists of an e-mail containing the title, authors, abstract, and possibly a significance statement and solicit their opinion.

The referees
Well, you have just found out that your manuscript has been sent out to review. So what is actually happening? Remember the “feelers” sent out above? If the lab head contacted feels that the work belongs in that journal, feels qualified to review the work, doesn’t have any conflicting interest, and has the time, then he or she will be asked to review the work. Now what is supposed to happen is that this scientist receives the manuscript and reviews it, keeping the information in strict confidence. Some lab heads do this, but some farm out the paper review to their senior graduate students and postdocs. You should keep this in mind – once you send the work out the door, the question isn’t “Does anybody in my field know about my work?”, but rather “How many people in the field know about my work?” Even if the lab head has farmed out some of the legwork of the review, almost all will read the manuscript themselves and come to their own conclusions.

When the referees submit their reviews, there are parts that are relayed back to the authors, and parts that only the editors see. In addition to a field where you can enter editor-only comments and concerns, there are also a series of statements that the referee must choose from. They are some form of the following:

• Publish without revision
• Publish with minor revisions
• Consider for publishing upon major revisions
• Reject

As the author, you never actually see what the referee recommended – you receive the recommendation of the editor. The editor’s recommendation is influenced by the referee’s recommendations, but the editor has the freedom to come to his or her own conclusions. It’s good to bear this in mind, because sometimes a referee will be held responsible for killing a manuscript, when he or she actually recommended publication (almost certainly with revisions) but the editor disagreed. (We’re not supposed to know who reviews our papers, of course, but speculation on referee identity is a favorite pastime of many scientists, and raised to the level of an art by some.)

Dealing with the reviews and writing your response

Your manuscript wasn’t rejected, but the referees did have some criticisms, though. Don’t worry about that – we all get them. I think criticisms are how scientists prove to others that they actually read something. Referee comments can fall into one of several different categories, though.

- The referee has no idea what is going on. Sometimes the editor just picks a bad referee, and the referee either didn’t want to admit ignorance or truly believes they can review papers outside of their discipline. The best example of this that I’ve heard was a friend that had a math-intensive paper reviewed by somebody that didn’t know what basic mathematical symbols were, mistaking them for variables which were left undefined, to their great outrage. When you get these types of comments (and you will, eventually), the hardest thing to do is to write a polite response, but you should. Remember, you don’t know who the referee is, but they know who you are, and scientists have excellent memories.

-The question that you already answered – in the manuscript. The referee probably just missed the fact that you explicitly addressed this question/issue. Or maybe you didn’t address the issue as clearly as you could have. Read over what you wrote again to make sure it’s clear, and if it is find a polite way to say “we already said that” in the response letter.
-You didn’t cite my favorite papers. If you have the space to add a couple sentences and/or a couple references, add them. This person spent their valuable time helping you publish your paper, so do as they ask. Chances are that you should have cited them in the first place, and citations are one criteria that is used to evaluate established scientists, so it isn’t just an ego thing. Now if they ask you to rewrite your entire introduction, it may be a different story.

-The nitty-gritty details question. These are usually asked by the people right in your field, and they can go one of two ways. Some are so specific and detailed that trying to incorporate the information that the reviewer requests into the manuscript itself is difficult to do while keeping the paper accessible to the average reader. In this case, it may suffice to just answer the question in the response letter and the referee will be satisfied that the work is solid. On the other hand, the issue may be a more important point than you originally thought, so if the information can be incorporated without confusing the average reader, incorporate it into the manuscript.

-This is nice, but I would rather have read your next paper. This one is tough – you’ve put together ~6 figures (and a bunch more in supplemental), and all the referee wants are the next 6 figures, which you thought would be your second paper. Re-read your title and conclusions – does your data solidly support these statements? Sometimes referees ask for the next paper because the authors promised more with the title than they delivered with the data. If this isn’t the case, then politely state in the response that you agree that the next 6 figures/experiments would be very interesting and exciting – so exciting that they will get their own paper.

-You did it all wrong. This is the toughest. The referee doesn’t like your reagents, your experimental design, your interpretation of the data, or all of the above. I think the most important thing to do is try to distance yourself from the experiments and ask if there is any validity to what they are saying. Keep an open mind and try doing the experiments their way and see what the results are. If the results are different, then they may have saved you from having to retract the paper in the future. If the results are the same, then your work and the conclusions have only gotten tighter. If their requests are completely off-base or unfeasible (the experiment that costs millions of dollars or uses an instrument with specifications that hasn’t been invented yet), then you need to communicate this politely in the response letter.

One thing that you should bear in mind when writing the response to the reviews is that the editor is obliged to communicate the referee’s review to you, regardless of whether the editor agreed with it or not. Your response may only be read the editor, or it may also be forwarded back to the referee for additional comment. This is why I emphasized politeness so many times above. You also have to strike some balance between rebuttals to a referee’s criticisms and changes to the manuscript. If the editor requests additional comments from the referee and all you have done is argue the validity of the referee’s comments without changing anything in the manuscript, then the referee isn’t going to be very happy about the situation. On the other hand, you should never weaken, diminish, or reduce the clarity of your manuscript just to appease every comment of a referee.

Page Proofs
You have survived the review gauntlet, and the journal has accepted your creation. You aren’t quite done yet, but don’t worry, the next step is pretty painless. After accepting your manuscript, the journal shipped it off… someplace… where they format it for publication. (I like to think it is a Keebler-esque tree-house full of elves with laptops rather than the cubical-farm that it likely is.) These formatted files are called the page proofs, and these files will show up unannounced in you inbox with a ~48 hour deadline for you and your mentor to review and return them. After you return them, that is it. That is what is published, warts and all. Fail to return them? They might publish the paper as-is. So pay attention to your inbox and the details!

Remember that list of words that your mentor hates? Well the journal/line editor has their own list, and they have edited your text. The best advice I was given is to read the paper backwards, one sentence at a time. This forces you to read what is written, and not to start reciting the manuscript you wrote in your head. This can allow you to catch any typos that have snuck through, and to catch the edits that the line editor has made. If the edits that have been made don’t change the meaning or clarity of the statement, let them go – learn to pick your battles. Don’t forget to check the references, either. It may be a good idea to bribe a grammar/spelling gifted friend to help you out, as well.

Scrutinize the figures both printed at actual size (preferably on several different printers) and enlarged on your computer screen. Has the resolution changed? Are the brightness and contrast acceptable? Are there any typos/misspelled words in the figure itself? This is your last chance to fix it in the manuscript without the potentially embarrassing publication of a correction.

Congratulations! You have published your first paper! Now how far are you along on the next manuscript?

Once you have written the first draft and handed it off to your mentor, the editing process begins. Depending on the personalities involved, this could be a very difficult time in the relationship between you and your mentor. Here are the perspectives (perhaps mantras?) that I try to maintain during the process.

It isn’t just your paper
After spending a lot of time working on the experiments, presenting lab meetings, and maybe even presenting a poster or giving a talk, you have come to think of the project as yours. It’s understandable, and perhaps even necessary to maintain the level of dedication required to bring many projects to fruition. With the edits of the first draft can come a hard truth – it isn’t just your paper. Your mentor thinks it is also, or even primarily, his or her paper. This isn’t an entirely unreasonable perspective, since in all likelihood the rest of your field will actually refer to the paper as “the new one out of Dr. Bigshot’s lab,” and your mentor will be held far more accountable for what is said and how it’s said than you will. Therefore, your mentor may have some very strong opinions on the exact wording of the manuscript.

Don’t take it personally
Everybody has their own way of saying something, and if your mentor has crossed your way out and written his or her way in, don’t take it as a personal criticism. It may well be that your phrasing was just fine, and they aren’t necessarily correcting what you wrote, but rather putting their own stamp on the manuscript. In this process you will discover that they have words they love to use, and words they hate to use. Over time, you’ll likely discover that you have similar lists of words.

In the last article in this series, I warned against spending a large amount of time and effort to fine tune a difficult passage in the first draft of a manuscript. Not only should you not do this because of time and effort considerations, but because spending that amount of time and effort makes you more invested in those areas of the manuscript. This makes changes or deletions of these sections much more frustrating to deal with.

In terms of a learning experience, one of the most difficult aspects of the editing process can be separating the changes due to stylistic differences from the changes made in the name of a more universal truth. Stylistic differences are just a matter of personal choice, and shouldn’t be taken too seriously, while the later changes are things you can learn from and use in the future. The easiest way to tell the difference is to go over the edits with your mentor. They will have a definitive reasons for the changes that you should probably learn something from while you’ll hear things like “I thought this sounded better” or “I just don’t care for that word” for the edits that come down to stylistic issues.

Sometimes things change
The line-editing isn’t necessarily the most frustrating element in the editing cycles. A number of times I’ve seen mentors get a manuscript full of experiments that they’ve seen over and over again, incorporated into a narrative that the student has discussed with them (and hopefully they approved of at the outline stage), and it wasn’t until they saw the whole package put together that they realized that they didn’t like the story. This can results in a major re-structuring of the manuscript, changing everything from the title on down. Sometimes it also means re-doing experiments to address subtly different questions than the original experiments addressed or including totally new experiments.

This can leave you with the feeling that a contract has been broken, and is usually expressed with sentences that begin “But you said that…” It undermines your confidence that the next version of the manuscript will be acceptable, as though you are trying to hit a moving target. I mean, you did everything they said to do with the first draft, and that wasn’t good enough, so why should you believe that doing what they say now will be acceptable either?

Now, I believe that this particular scenario happens less often with good mentors, who are engaged with their students and actively thinking about their projects long before it’s time to write the paper. But even with really good mentors, you have to realize that sometimes things change. Sometimes when everything is packaged together, you realize that you need one more experiment to prove the model you’ve put at the end of the paper. Or, after talking to a colleague at a meeting, your mentor thinks that the field would be more receptive to a closely related, but slightly different focus of the paper. There never was a contract between you and your mentor that said “I will do these ten experiments, and you must publish them without asking for any more.” Try to keep an open mind and see how the new requests will make the manuscript better.

You and your mentor are on the same side
In these situations, it’s critical to try to maintain a healthy perspective, like the one above. My own rule is to look at each change that’s made and ask “Is the new version incorrect or misleading in any way?” If the answer is no, then accept it. If it’s yes, then set it aside and discuss your concern with your mentor. Take big deep breaths when you need to, and set time aside in your schedule to do relaxing activities if things start getting under your skin. Look for the humor in the fact that in the second round of editing, your mentor is quite likely to re-edit their own work from the first round.

And remember each round of edits gets you closer to publishing your first paper, and each paper gets you closer to graduating.

Another advantage of working in biotech is the experience you gain on business as a whole. You learn valuable skills such as marketing, production, QC processes, operations, technical manual writing, and even a little bit of sales.  And another big plus is that you have the ability to move around the company, try new jobs, and move up the ladder to a position of leadership in an organization, if you have the drive and desire.

So now you’re thinking that sounds pretty good, right? And you’re wondering if you would be a good fit for biotech science? To help you figure if you would be happy in a biotech science lab, here are some questions I came up with that you can ask yourself. If you say “yes” to all of these, then you may want to consider working in biotech.

1. Are you outgoing and enthusiastic?

Companies need scientists who are not afraid to speak up in a room of people and voice an opinion. A scientist who enjoys talking to the customers and helping them is a huge asset to a company. Enthusiasm is also important in keeping the morale of a team upbeat. And it also relates to a person who thinks positively, which is a must when trying to meet a deadline for a product launch.  If you don’t have confidence in your ability to figure something out, you probably won’t.

2. Do you work well in teams?

Making a new product is a team effort. It requires the input of operations, production, marketing and R&D. No product is a success based on the efforts of a single person. A person that cannot get along with others or makes it difficult for the rest of the team to do their jobs will not be a good fit in biotech. The ability to work in teams is a must.

3. Can you handle change?

In biotech, the only thing you can count on being the same is change. It might be changing your desk (I had to pack up and unpack my cubicle four times in one year), changes to the team you work with, changes to procedures to follow that make your job more tedious instead of saving you time, and big changes such as changes in who you report too.  If you prefer the status quo and like consistency, you may not be a good fit in biotech.

4. Do you like to invent things?

Have you ever wanted to be on a patent? Do you like to think up new ways to do something that no one has ever thought of before? You’ll have a chance to do that in biotech. Depending on your job, you may have the chance to be inventive in between running assays, or your whole job might just be  coming up with new trade secrets or patentable inventions.

5. Do you like to travel?

There is ample opportunity to get out of the lab and go to conferences or visit customers if you desire. Marketing managers love it when scientists want to present their work in posters and spend some brief time in the booth greeting researchers.  Unlike academics where travel may be limited to one meeting a year, in biotech, you can go to all of the big conferences or spend time visiting customer labs to help them with experiments when needed. Another role in a biotech company is called a Field Application Scientist (FAS). This person’s whole job is to travel to scientists’ labs and help people personally with the companies products or robotics.

6. Can you handle pressure?

Obviously there is pressure at all jobs and certainly academic life has it’s own unique pressures and stress. In biotech, the pressure can become difficult because hundreds of thousands, maybe even millions of dollars can be on the table, riding on your success. As the lead R&D scientist for a product with a projected revenue in the millions, there is pressure for you to produce a flawless product from the entire team: marketing managers, top level executives, sales people, and even within the team.  Pressure also comes from too tight deadlines and problems staying on track when the research plan is set up by people other than you. When you have to tell marketing that a product launch is going to be late because something is not working, expect a storm. These are people who are not scientists so do not always have a concept of how science works and how things don’t always go according to plan.

7. Are you ok with public speaking?

Biotech science requires an outgoing personality. Since you are working in teams, you’ll need to have the confidence to speak up. And if you like to travel, you will be doing a lot of handshaking, networking, and presentations, so being comfortable in groups is important.

8. Can you multitask?

Not everyone likes to do more than one project at a time. Or handle more than one issue at a time. In biotech, this will be a must. Most people have to handle more than they are normally comfortable with so the ability to manage mulitple projects and not make mistakes will be a huge asset. If you enjoy having your plate always very full and like variety in your work, then you’ll enjoy biotech life.

9.  Do you learn new methods quickly?

In biotech, you’ll need to get up the learning curve very quickly. Everyone is moving at a fast pace and in some companies, people do not have the time or patience to help new people. Everyone is multi-tasking, remember. The ideal scientist in a biotech can read a protocol and get it down pretty quick. They can get comfortable in a new surrounding quickly and get to working on the project assigned without needing a lot of training. All new people need training, of course. But a strong technical background with a lot of techniques under your belt will serve you well in a biotech company.

10. Do you have an entreprenurial spirit?

Working in a biotech company, no matter the size, requires that your mind is centered on the goals of the company: to make money.  If you have ever thought about starting your own company and want to be your own boss, or if you enjoy the challenge of developing a method and watching it’s commercial success, you’ll enjoy working in biotech.  Afterall, the success of a company is measured on sales so you have to be okay with making profit off your hard work.  It would be nice to be able to develop a life saving method or product and give it away for free. However the livelihood of many employees at a company depends on the scientists coming up with exciting and new product ideas that people want or need.  And we have to give the sales reps something to sell so they have jobs too, right?

Let me know what you think and if you have anything else to add. I am sure there are a few biotech scientists reading so share you experiences of life in biotech for our academic scientist friends. We welcome your feedback.

You have been pounding away at your project, probably for a year… or two… or three… Anyhow, you now have a collection of figures that seem to tell quite a nice story, and now it’s time to write the first draft. There has been a lot written on the mechanics of scientific writing, and even if you haven’t read that material, you have read a lot of papers. So rather than giving you a section-by-section breakdown on paper writing, I’m going to outline my process for generating the first draft of my manuscripts.

Create an outline
Opinions differ on this point, but I personally like starting things off with an outline. This allows me to map out the order in which I introduce material, describe the experiments, and list the key points that I want to make about each without getting wrapped up in phrasing and transitions. I do the whole thing – Introduction through Discussion including the Experimental Procedures. Usually at this stage I can see problems that might arise in the presentation and fix them without deleting sentences I spent hours crafting. It also serves as my ‘map’ while writing to keep me focused and aware of where I’m going in the narrative.

After finishing a reasonably detailed outline, it would be a good idea to give it to your mentor along with the figures (complete with legends) to look over. Hopefully, your mentor can give this some serious consideration, and afterward meet with you and discuss it. If there are large, sweeping changes to the order of the experiments, or if he or she envisioned a different focus of the work than the one you’ve put forward, then the changes can be made here where the time investment in the prose is still minimal.

Determine the destination
You may have discussed this with your mentor before now, but if you haven’t, then the meeting where you discuss the outline would be the best time to talk about what journal you will be sending it to. This becomes most critical at the top of the pile – the manuscript you write for Nature will be radically different from the one you write for Cell. Other journals may not have such extremes in the structure, but may have other content requirements that should be kept in mind. For example, a primarily biochemical paper intended for The Journal of Biological Chemistry will likely have to have more emphasis on biological relevance than if the same manuscript were being sent to Biochemistry.

Just spit it out
Now you have the outline with your mentor’s tentative stamp of approval and a solid idea about where it should go, it’s time to start fleshing it all out into a paper. My next piece of advice is to be careful how much time you spend getting the wording just perfect on any particular section. If you are having a tough time saying something just right, then take your best stab at it and leave behind an e-note saying “I’m not certain I’m happy with this yet.”

On one occasion I spent three days writing and re-writing just one paragraph that I thought was essential to the paper. In the first round of edits, my mentor eliminated the whole thing. That was three wasted days, and in the end the paragraph wasn’t as necessary as I was convinced it was at the time. When you find yourself at a loss for just the right words, do your best in a reasonable period of time, mark it, and move on. The statement may not be as necessary as you think or your mentor may have just the right turn of phrase needed to make the point.

Let it rest
You just finished writing the first draft! You even referenced it! Now, if time allows, close the file and don’t open it again for a week. You need time for your mind to ‘reset’ on the subject so you can read what you wrote again with fresh eyes. Besides, you probably should go re-read the papers you referenced again, just to make sure they said everything you thought they said when you cited them. If you haven’t written the Experimental Procedures yet, this is also a good time for that.

Once you have let the manuscript rest, come back to it and read it again, slowly. Does it ‘sound’ like the published papers you’ve been reading? (In other words, did you get the tone right?) Make sure the narrative flows, fix the typos, and correct any wrong words (there, they’re, and their…). Of course, if you find any larger issues with the science, fix those as well.

Phone a friend
If you have a senior labmate with some publication experience that can be bribed with either cookies or beer, then have him or her read the manuscript over quickly and get some feedback. Now may not be the time for multiple opinions – I would ask just one person that you trust and respect as long as it doesn’t offend anybody else. Stress that you aren’t looking for line-editing, just general impressions of your writing style and the storyline. Once you get their feedback, try to incorporate it.

Now you have your first draft. It’s time to give it to your mentor. The next step – surviving the editing process.

John’s comment on Jode’s recent article here on Bitesize Bio: “Good idea on marking the rotor for 3 tubes Jode. One of those tiny (perhaps tragic) pleasures is when you drop the 3 tubes in quickly and get in spaced perfectly first time. Because usually its drop them in and then move one tube 1 space over and then 1 other tube 1 space back.” made me laugh in recognition.

How many of us can identify with that small surge of pleasure when something tiny and insignificant goes right in the lab?  Here are a few things that will make me smile any day:

• getting tubes balanced in the microfuge on the first try (so right, John!)

• weighing out a sample to exactly 20.00 g on the balance

• having just enough paper in the printer to print out an article

• entering sample 1000 in the -80°C database

• grabbing the right number of tubes for all your samples without counting

• ejecting all 8 tips from the multichannel pipettor at the same time

• finding the microscope already set to the right objective and filter

• having enough different Sharpie colors to label all your different samples

• dividing a piece of tinfoil exactly to cover bottles for autoclaving, with no scraps left over

• filling up every well of an agarose gel

What tiny pleasures in the lab can make your day?