Why is the observed Western Blot band size different from predicted size?

2012-11-09T09:19:00.000-05:00

The predicted M.W. is based on protein sequence analysis; however, some factors might lead to an observed band size that is different from the predicted size. The reasons might include:

1.Post-translational modification (PTM):
a. Some post-translational modifications might lead to increased protein size, including
phosphorylation, acetylation, methylation, glycosylation, sumoylation, ubiquitination,
etc.
b. Some post-translational modifications might lead to decreased protein size including
phosphatidylethanolamine conjunction (e.g. LC3-II)
c. Some proteins may be cleaved to form an active or mature form; this process will
lead to a decreased protein size (e.g. Notch activation, Caspase activation, etc.)
d. Some websites provide useful PTM information

i. HPRD http://www.hprd.org/
ii. ProSite http://www.expasy.org/prosite/
iii. ELM http://elm.eu.org/
iv. CBS data sets http://www.cbs.dtu.dk/databases/
v. CBS prediction Servers http://www.cbs.dtu.dk/services/

2.mRNA splice variants (Isoforms):
Through alternative splicing, one gene can generate different proteins with different M.W. Regulation of alternative splicing depends upon cell type, conditions, etc.

3.Multimerization:
Some proteins could form dimers or multimers, increasing the M.W. This phenomenon usually can be found in reducing gel condition; however, strong interactions may still be seen with higher molecular weight proteins even in denaturing gel.

4.Protein charge:
The observed size could also potentially be influenced by the protein charge

5.Different species:
Different species likely have different protein sequence and PTM, which can lead to a different protein M.W.

Source: http://www.genetex.com/WebPage/Product/PredictedTarget.aspx

Trypan Blue

2012-02-08T13:49:00.000-05:00

Trypan blue is a vital stain used to selectively colour dead tissues or cells blue. It is a diazo dye. Live cells or tissues with intact cell membranes are not coloured. Since cells are very selective in the compounds that pass through the membrane, in a viable cell trypan blue is not absorbed; however, it traverses the membrane in a dead cell. Hence, dead cells are shown as a distinctive blue colour under a microscope. Since live cells are excluded from staining, this staining method is also described as a dye exclusion method. (from:Wikipedia)

Viable Cell Counts Using Trypan Blue (Invitrogen)

Trypan Blue is a vital dye. The reactivity of trypan blue is based on the fact that the chromopore is negatively charged and does not interact with the cell unless the membrane is damaged. Therefore, all the cells which exclude the dye are viable.

PROCEDURE: Trypan Blue Staining of Cells

1. Place 0.5 ml of a suitable cell suspension (dilute cells in complete medium without serum to an approximate concentration of 1 x 10^5 to 2 x 10^5 cells per ml) in a screw cap test tube.

2. Add 0.1 ml of 0.4% Trypan Blue Stain. Mix thoroughly.

3. Allow to stand 5 min at 15 to 30°C (room temperature).

4. Fill a hemocytometer as for cell counting.

5. Under a microscope, observe if non-viable are stained and viable cells excluded the stain.

Reference: 1. Freshney, R. (1987) Culture of Animal Cells: A Manual of Basic Technique, p. 117, Alan R. Liss, Inc., New York.

Along with Cell

Why is the observed Western Blot band size different from predicted size?

Trypan Blue