Wednesday, March 14, 2012

Western blot biases

I think I have this one right.

Western Blot kits are either store bought or produced internally. These difference seem to lead to preferential expression of bands ostensibly related to variable degrees of antigenic expression.

Clongen and Labcorp (using the same kit) express more 23 bands.

Stony Brook expresses more 93 bands

IgeneX expresses more 30,31 bands.

Perhaps they are all correct.

I have found this helpful in my efforts to reconcile the different reports.

4 comments:

worriedmom said...

Are there any reference standards against which to check the kit or in-house preparations?

Thanks for doing these blogs!

Pearl said...

I womder if they use different strains in their tests.

Doesn't differing results from differing labs prove that denying treatment because of one negative result makes no sense?

Sarah Hengel said...

So this interesting, but as a graduate student who runs western blots, the variability in the number of bands on the western can come from many things.

First, you might see more bands from one westernblot compared to the other due to an increase time of transfer from the gel to the PVDF membrane. The proteins isolated from blood are run into the gel and transferred to the membrane.

Secondly, the purity of the antibodies can be in question. Testing of the antibody can indicate if some bands are non-specific etc.

Thirdly, I would expect to observe different protein bands on different days. Protein expression changes when you get a cold, when you get a pin prick in your finger, when you get a bruise, therefore I am not surprised that the number of bands changes.

If you assume that the antibody is pure, then If you see the band, the protein is there.

Sarah Hengel said...

Although this does not give any quantitative data unless proper controls are used...