Blog | Abcam

Dipstick enzyme activity and sandwich ELISA kits video

Tue, 07 Feb 2012 13:17:00 -0000

Ideal for situations where a rapid, simple and sensitive assay solution is desired for quantitative analyses

Advantages of dipstick assays

This technique requires minimal sample preparation and few overall steps. The total assay time is less than one hour and requires as little as 3µg of sample material. It is suitable for a wide range of sample types: such as tissue, cultured cells, blood and cheek swab material and can be used in human, mouse and rats species and does not require isolation of mitochondria. There are corresponding quantitative sandwich ELISA assays for most enzyme activity assays and combining the two, it is possible to generate specific activity data.

Dipstick enzyme activity assays apply a novel approach, whereby target proteins are first immunocaptured from tissue or cell samples before subsequent functional analysis. Dipstick assays use the well-established lateral flow concept, whereby capture antibodies are striped onto nitrocellulose membrane and a Whatman paper wicking pad draws the sample through the antibody bands. Detector antibodies, conjugated to gold, are dried in the wells of a 96-well plate. Sample is added to the well, the dipstick inserted and within minutes the line for each target is revealed as the protein-detector antibody-gold complex binds with the capture antibodies. A positive control goat anti-mouse antibody line is included on all assays to ensure that adequate wicking of the sample occurs. Assay are also available in a multiplex format for multiple target proteins.

Additional flow cytometry troubleshooting tips

Mon, 23 Jan 2012 13:37:00 -0000

Related resources

Our Scientific Support team share some additional flow cytometry troubleshooting tips from a recent workshop.

In order to better serve our customers, the members of the Abcam scientific support team frequently attend training sessions that increase and refresh our technical knowledge. In October, Elena, Jackie, and Caitlin from the US team completed a day-long flow cytometry workshop at the Flow Cytometry Core of Beth Israel Deaconess Medical Center in Boston, under the instruction of technical director John Tigges. The day started with a brief history of fluorescence and flow cytometry (the first cytometer was invented in 1968!) and continued with in-depth training on the fluidics, optics, and electronics systems involved in the machines. John was kind enough to also give us a full afternoon running fluorescent beads on an LSRII!

What we learned

When immunostaining for proteins that will be rare in the cell population, use a stronger fluorophore like phycoerythrin (PE).
Don't change any voltages once they're set to the unstained control.
If the sample pressure is too high, multiple cells will be analyzed at once. This is fine for IF analysis, but if the CV's are important (such as for cell cycle or ploidy studies) then this needs to be corrected.
Prevent air from being introduced into the system, otherwise the pressure won't be sufficient and events won't be seen.

» For more tips, visit our previous blog article: Top 10 tips for flow cytometry

Latest Abraffle winner

Wed, 11 Jan 2012 23:17:00 -0000

Here are the latest Abraffle winners for the month of January, 2012, from our prize draws at tradeshows and other events. Abraffle winners all win a free antibody of their choice!

Winner: Dolores Russo – PUC
Event: Multiple Perspectives to Brain Development – Santiago, Chile

Winner: Cecilia Opazo - U Talca IBUB
Event: VI Reunión de Biologia Vegetal - Pucón, Chile

Please email, within two weeks of January 11, 2012, to daniela.hodgkins@abcam.com from the email address you submitted in order to claim your prize.

Abcam organizes our own conferences to provide opportunities for junior scientists to showcase their work, we send our scientific experts all over the world to keep up-to-date with the latest research. You can also view the scientific tradeshows that we will be attending, we look forward to meeting you!

The Abcam Calendar 2012

Thu, 05 Jan 2012 12:08:00 -0000

Welcome to the Molly Cup!

This year Molly is putting on her running shoes, squeezing into her leotard and sharpening her javelin in readiness for the Molly Cup. Compete to win monthly prizes and get your name up in lights on the leaderboard!

Every month, throughout the year, we will be releasing a new game to play. Take part in all 12 events for your chance to win the Molly Cup (and even more prizes). 2012 calendars will be distributed in shipments until stocks last. You might also be able to pick one up if you come and see us at a tradeshow or one of our events.

Play the January game now

Winners of Abcam’s building blocks competition announced!

Mon, 19 Dec 2011 18:40:00 -0000

It all started, when out of the blue, a few of our customers submitted some of their creations made from our building blocks. Wow, what a creative bunch we thought! This gave us the idea to run a competition to uncover some more of your hidden talents.

We were astounded at the response we received. It was extremely difficult to shortlist the 15 finalists, so we breathed a sigh of relief when round two of the competition went to the public vote.

We are pleased to announce that you chose Minion as the winner, with Abtron and Fish second and third respectively. The first prize winner will receive $5000 Abcam credit, the second place winner will receive $1000 lab retreat and the third prize winner will receive a $500 gift basket.

Thank you to everyone who has taken an interest in the competition. Check out our Facebook page to see all the finalist entries.

*Winners will be contacted directly about claiming their prizes

The Abcam building blocks competition: Who’s got the X factor? You decide...

Mon, 28 Nov 2011 13:43:00 -0000

Vote now

There will be a prize for 1st, 2nd and 3rd highest voted images:

1st prize:
$5000 Abcam credit

2nd prize:
$1000 lab retreat

3rd prize:
$500 gift basket

With such a high standard of entries received, it was always going to be a difficult task to select the finalists for round two of our competition. After much discussion, deliberation and debate, we have shortlisted 15 finalists to face the public vote.

How to place your vote

Simply go to our Facebook page and browse the gallery of finalists
Select the Like button for the image that you think is most worthy of winning one of the great prizes on offer

We would like say a big thank you to all competition entrants. We were so impressed with the images received, we will be creating an art gallery of all submitted entries on our Facebook page for everyone to enjoy once the competition closes.

Terms and conditions

Voting will close on Friday December 16, 2011. Votes received after this date will not be counted
Voters can 'like' more than one image
The three entries to receive the most votes will be declared as winners of the competition
Winners will be allocated either the 1st, 2nd and 3rd prizes according to the number of votes received for their entry
If equal numbers of votes are received, the Abcam judging panel will make a decision as to who should be named the winner. The judge's decision will be final
Winning entries will be announced on the Abcam Facebook page and blog on Monday December 19, 2011

Abcam's scientific roadshow in Germany, 2011

Wed, 23 Nov 2011 11:05:00 -0000

Related resources

Join us for our scientific roadshow and learn more about how to optimize your ELISA, western blot, IHC and ChIP experiments.

Our experienced scientific support specialized will give you tips and tricks, help you troubleshoot your experiments and answer your questions. Furthermore she will explain more about our 100% testing offer, the upcoming webinars and why Abreviews are important for your own research.

We welcome you to our FREE seminar on laboratory techniques in the following cities:

Berlin: November 28, 2011

1. Session

Time: 10h - 11.30h
Where: Max-Delbrück-Centre - small lecture hall Axon 2

2. Session

Time: 13.30h - 15.30h
Where: MPI for molecular genetics - seminary room in the main building

Hannover: November 29, 2011

1. Session

Time: 10h - 12h
Where: MHH Hannover - building J-2 - lecture hall E

2. Session

Time: 13h - 15h
Where: TiHo Hannover - library department of parasitology

Göttingen: November 30, 2011

1. Session

Time: 10h - 12h
Where: ENI Göttingen -seminary room ground floor R. 0.055

2. Session

Time: 13.30h - 15h
Where: MPI for experimental medicine - seminary room A1

We look forward to seeing you soon!

Immunoprecipitation: Procedures, pitfalls and protocol tips

Mon, 21 Nov 2011 11:18:00 -0000

"I just want to thank you all again for this webinar. It was very helpful and much appreciated".

— Webinar attendee

For questions about future webinars email: events@abcam.com

Watch another of our popular webinars for free, 'Immunoprecipitation: Procedures, pitfalls and protocol tips'.

Immunoprecipitation is a method that enables the purification of a protein or protein complex. Proteins are physically isolated from a biological sample and subject to further analysis. IP can be used for a wide variety of applications such as protein-protein interactions, post-translational modifications, protein function, and expression profiling. Thus, this technique is a powerful tool for researchers studying cancer, development, cell signaling pathways, and disease. It also makes this an important tool to researchers studying biological process. Please view this webinar for a presentation on Immunoprecipitation procedures, pitfalls, and protocol tips.

Latest Abraffle winner

Wed, 09 Nov 2011 21:37:00 -0000

Here is the latest Abraffle winner for the month of November, 2011, from our prize draws at tradeshows and other events. Abraffle winners all win a free antibody of their choice!

Winner: Jennifer Yu
Event: Cleveland Clinic Vendor Show

Please email, within two weeks of November 9th, 2011, to Abraffle@abcam.com from the email address you submitted in order to claim your prize.

Latest Abraffle winners: Week commencing November 7, 2011

Tue, 08 Nov 2011 12:48:00 -0000

Here are the latest Abraffle winners for week commencing November 7, 2011, from our prize draws at tradeshows and other events. Abraffle winners all win a free antibody of their choice!

Winner: Heather Zimmermann
Event: University of Washington, October vendor show

Winner: Christina Ironside
Event: Fred Hutchinson Cancer Research Center, October vendor show

Please email within two weeks of November 8, 2011 to Abraffle@abcam.com from the email address you submitted in order to claim your prize.

Abraffle winner: October 2011

Fri, 04 Nov 2011 14:15:00 -0000

Here is the latest Abraffle winner for the month of October, 2011, from our prize draws at tradeshows and other events. Abraffle winners all win a free antibody of their choice!

Winner: Dr. Monica Lamas
Event: Cell Signaling Networks meeting in Merida, Mexico

Please email, within two weeks of November 4th, 2011, to abraffle@abcam.com from the email address you submitted in order to claim your prize.

Ten tips for successful flow cytometry results

Thu, 03 Nov 2011 11:34:00 -0000

Top 10 tips

Sample preparation
Fixation
Lasers alignment
Use the correct controls to set up the instrument
Event rate
Gating strategy
Secreted proteins
Multicolor analysis and compensation
High background
No staining

Related resources

Flow cytometry webinar

Our Scientific Support flow cytometry experts have put together a top 10 list of things to consider to help produce successful results from your flow cytometry experiments. We hope you will find these useful.

1. Sample preparation

Generally, each sample should contain between 1 X 105 to 1 X 106 cells /ml in 100 µl in PBS containing 1% BSA or serum.

Keep the samples on ice at all times during preparation and staining, and at 4^oC for storage after staining. Ensure a single cell suspension to enable accurate data and to ensure the fluidics system does not block. Cell suspensions can be filtered to remove clumps, e.g. use a 50 µm Nylon Mesh, and vortex immediately before running on the flow cytometer.

Blood samples:

Use fresh samples collected in tubes containing anticoagulant and transferred to the laboratory on ice. Refer to the supplier regarding the type of collection tube and anticoagulant for certain experimental settings (EDTA, Citrate, Heparin).

There are several commercially available red blood cell lysis buffers, and we would recommend to follow the protocols provided by the manufacturer. Alternatively, red blood cells can be removed from the sample using the following commonly used red blood cell lysis buffer (prepare fresh):

KHCO3 10 mM
NH4Cl 150 mM
EDTA (pH8) 0.1 mM

Tissue samples: Use enzymatic methods or a homogeniser to create a single cell suspension. Cell culture: Harvest cells when they are about 70 – 80% confluent to ensure they are viable. We recommend avoiding samples that are over confluent and/or may contain too many dead cells or debris. Do a cell count of the single cell suspension using trypan blue.

»view our cell count protocol

For all sample types, ensure cells are >95% viable before staining. This will help to ensure the results are accurate and that there is as little debris as possible.

2. Fixation

Depending on the sample type, sample may need to be fixed post staining.

Membrane proteins:

Samples are usually fixed after antibody staining with 0.1 to 4% PFA for 10 minutes to overnight. If they are not fixed, they will need to be run through the flow cytometer to collect data immediately after staining.

Intracellular proteins:

Permeabilization is required for intracellular staining, to allow access of the antibody into the cell, and the cells must be fixed before they are permeabilised. For sample, fix with 0.1% PFA for 10 minutes, then permeabilise with 0.1% Saponin for 10 minutes. Or when using acetone or methanol to fix (10 minutes ice cold), these solvents will both fix and permeabilise. (Note that acetone can sometimes increase autofluoresence and adjustment of settings may be required).

Note: Fixation can change the FCS/SSC light scatter properties of the cells

Health and safety: Unfixed cells may expose the operator to additional biological hazards and we would recommend to follow the correct health and safety guidelines for handling the samples. For more information on fixation and permeabilization view our Intracellular staining protocol.

3. Lasers alignment

Ensure lasers on the flow cytometer are aligned correctly by running flow check beads and/or single color control beads or samples, and adjusting alignment if necessary. If the lasers do not align correctly or if drift occurs, you may need to consider having the machine serviced.

4. Use the correct controls to set up the instrument

Use the positive, negative (or isotype) controls to set up the flow cytometer correctly. Other controls required are listed on our 'recommended controls' flow cytometry webpage:

5. Event rate

The flow rate is usually set around 50 µl/min. Most flow cytometers have 'low', 'medium' and 'high' flow rate settings to select from. Some machines will have an upper limit. If the event rate is very high, even when the flow rate is low, try diluting the sample in some buffer. Try to standardize flow speed and event rate for each sample.

Notes on the flow rate and event rate:

Data is more accurate if the sample speed is slightly slower Lower flow rate is used when greater resolution is required, e.g. for DNA analysis. The data will be more accurate if the samples speed is slightly slower.

Higher flow rate can be used for qualitative measurements e.g. immunophenotyping. In this case, data is less 'resolved/ as the cells are not so well aligned to the laser. But the data can be acquired more quickly.

6. Gating strategy

Gating allows the user to select regions of cells or a cell population. Gating is dependent on experimental settings and the information required from the data. New software now available allows for various gating shapes, and we would recommend to review the software instructions for further information on your machine.

Gating tips:

The first gate should always be set on the correct cell population in the SSC/FCS plot.
Regions drawn on negative populations (excluding scatter and viability gates) need to be pushed to the very edge of the x y axes to avoid excluding negative events that are crushed up against the axis.
Data follows a biological distribution that does not always fit with rigid gating. Be consistent and logical in how you draw regions, to minimize subjective bias for inter experiment comparison. We would recommend where possible to run all the samples from one set of experiments using the same machine settings and gates to ensure standardization.

7. Secreted proteins

Detection of secreted proteins is difficult as the protein will be released from the cell before detection, or may degrade rapidly. A frequently used solution is to block the secretion using a Golgi Block such as Brefeldin A and other compounds. Cells can incubated for 4 – 16 hours with Brefeldin A which prevents proteins being released from the golgi. The exact experimental conditions and time will need to be optimized. Any cells expressing the protein can then be detected. In this case, fix and permeabilization is required before staining to ensure the protein remains intracellular.

8. Multicolor analysis and compensation

When using multicolor analysis using different fluorochromes, emission spectra can overlap, fluorescence from more than one fluorochrome may be detected within one channel. Take care to select fluorochromes whose excitation and emission spectra overlap as little as possible. Check that the correct appropriate filters are selected and data is compensated for accurate results. This ensures that the fluorescence detected in a particular detector derives solely from the fluorochrome that is being measured. More information on compensation can be found on the following protocols page from our website:

Fluorescence compensation in flow cytometry
View our guide to fluorochomes, including a list of excitation and emission wavelengths.

9. High background

Sample line not clean

The sample line may contain debris, or some dyes such as PI can be 'sticky' and remain in the sample line. Try washing the machine through on a clean cycle. You may need to run the clean cycle several times.

Flow cytometer settings not correct

Use the positive control to try setting up the flow cytometer correctly again.

Excess antibody

Decrease the antibody concentration. You can also add detergent (e.g. 0.05% Tween 20) to the wash buffers to ensure washing away of excess antibody.

Dead cells and debris

Ensure cells are 95% viable before staining and that the sample does not contain too much debris. The threshold can be adjusted to remove debris from the histogram, but the threshold should not be set too high as the data will then be inaccurate. It is better to start again with fresh samples if possible, rather than use a sample containing a lot of debris.

10. No staining

Signal not correctly compensated

Check positive single color control is set up correctly on flow cytometer and gated/compensated correctly to capture all the events.

Insufficient antibody present for detection

Increase amount/concentration of antibody.

Intracellular target/epitope not accessible

Check if target protein is intracellular. For internal staining, ensure adequate permeabilization. To prevent internalization of cell surface proteins, everything must be done on ice or at 4°C, with ice cold reagents, to stop all reactions. For staining of cell lines, trypsin can often induce internalization and degradation of cell surface proteins, and more gentle detachment methods may be required.

The antibodies to some membrane proteins may detect an epitope in the cytoplasmic region of the protein, in which case gentle permeablization may be required.

Intracellular staining – fluorochrome conjugate too large

Fluorochromes for intracellular staining experiments should have low molecular weight. Large molecular weight flourochromes can reduce antibody motility and possibly its entry into the cell. For example, tandem dyes will be larger.

Machine settings

Use the correct controls (see point 4) to ensure the machine is set correctly.

Target protein not present/expressed at low level

Ensure tissue/cell type expresses target protein and that it is present in a high enough amount to detect.

Soluble/secreted target protein

Is the target protein soluble and secreted from the cell? It needs to be membrane bound or cytoplasmic to be detected easily by flow cytometry. A golgi-block step, such as with Brefaldin A, may improve the signal achieved for intracellular staining.

Fluorochrome fluorescence has faded

Antibody may have been kept for too long or left out in the light. Fresh antibody will be required. The primary antibody and the secondary antibody are not compatible Use secondary antibody that was raised against the species in which the primary was raised (e.g. primary is raised in rabbit, use anti-rabbit secondary).

Latest Abraffle winner

Fri, 28 Oct 2011 21:57:00 -0000

Here is the latest Abraffle winner for the month of October 2011, from our prize draws at tradeshows and other events. Abraffle winners all win a free antibody of their choice!

Winner: Erica Stein
Event: NIH Research Festival

Please email, within two weeks of October 28, 2011, to Abraffle@abcam.com from the email address you submitted in order to claim your prize.

Latest Abraffle winner

Wed, 26 Oct 2011 20:28:00 -0000

Here are the latest Abraffle winners for the month of October 2011, from our prize draws at tradeshows and other events. Abraffle winners all win a free antibody of their choice!

Winner: Kellie Machlus
Event: Longwood Galleria Annual Fall Bioshow

Winner: Genri Kawahara
Event: Center for Life Science-Life Science Product Show

Please email, within two weeks of October 27, 2011, to Abraffle@abcam.com from the email address you submitted in order to claim your prize.

Flow cytometry webinar, September 2011

Fri, 21 Oct 2011 15:47:00 -0000

"Our lab enjoyed listening to Ina's talk especially regarding the troubleshooting staining techniques"

— Webinar attendee

For questions about future webinars email: events@abcam.com

Related products:

Related protocols:

In September, 2011 Abcam held its second webinar focused on flow cytometry. You can now watch this webinar for free!

Flow Cytometry is a powerful technique that allows rapid and quantitative measurements of multiple parameter on individual cells simultaneously. Sheath fluid is used to transport these cells in single file past optical and electronic sensors to detect physical and biological characteristics. Therefore, multiparameter flow cytometry permits precise characterization of multiple cell subpopulations in complex mixtures. Flow cytometry has become an increasingly widespread technique in scientific research and can be used in a wide range of applications such as cellular phenotyping, apoptosis or cell cycle analysis.

Blog | Abcam

Dipstick enzyme activity and sandwich ELISA kits video

Ideal for situations where a rapid, simple and sensitive assay solution is desired for quantitative analyses

Related links:

Advantages of dipstick assays

Additional flow cytometry troubleshooting tips

Related resources

Our Scientific Support team share some additional flow cytometry troubleshooting tips from a recent workshop.

Latest Abraffle winner

The Abcam Calendar 2012

Welcome to the Molly Cup!

Winners of Abcam’s building blocks competition announced!

The Abcam building blocks competition: Who’s got the X factor? You decide...

There will be a prize for 1st, 2nd and 3rd highest voted images:

How to place your vote

Terms and conditions

Abcam's scientific roadshow in Germany, 2011

Related resources

Join us for our scientific roadshow and learn more about how to optimize your ELISA, western blot, IHC and ChIP experiments.

Berlin: November 28, 2011

Hannover: November 29, 2011

Göttingen: November 30, 2011

Immunoprecipitation: Procedures, pitfalls and protocol tips

Related products:

Watch another of our popular webinars for free, 'Immunoprecipitation: Procedures, pitfalls and protocol tips'.

Latest Abraffle winner

Latest Abraffle winners: Week commencing November 7, 2011

Abraffle winner: October 2011

Ten tips for successful flow cytometry results

Top 10 tips

Related resources

1. Sample preparation

Blood samples:

2. Fixation

Membrane proteins:

Intracellular proteins:

3. Lasers alignment

4. Use the correct controls to set up the instrument

5. Event rate

6. Gating strategy

7. Secreted proteins

8. Multicolor analysis and compensation

9. High background

10. No staining

Latest Abraffle winner

Latest Abraffle winner

Flow cytometry webinar, September 2011

In September, 2011 Abcam held its second webinar focused on flow cytometry. You can now watch this webinar for free!