Anti-Bad antibody (ab31310)

I am seeing a band between 20 and 26 kD on my Western blots, similar to the size seen on the image on your datasheet. But the predicted size is 18kD. What is the reason for this discepancy?

ANSWER:

Thank you for your enquiry.

The following paper has Western blot images of Bad running heavier than 18kD:

Jiping Zha,1 Hisashi Harada,1 Elizabeth Yang,1 Jennifer Jockel,1 and Stanley J Korsmeyer1; Serine Phosphorylation of Death Agonist BAD in Response to Survival Factor Results in Binding to 14-3-3 Not BCL-XL. Cell, Vol 87, 619-628, 15 November 1996

The reason why Bad runs at this weight on Western blots is unclear. We have a link on our datasheet for ab31310 in the caption of the WB image (and on datasheets of other antibodies where there is a discrepancy between predicted and observed MW) that leads to a discussion of possible explanations:

Why is the actual band size different from the predicted?

Western blotting is a technique that separates proteins based on size - in general, the smaller the protein the faster it migrates through the gel. However, migration is also affected by other factors and so the actual band size observed may differ from that predicted. Common factors include...

post-translational modification - e.g. phosphorylation, glycosylation etc which increases the size of the protein.

post-translation cleavage - e.g. many proteins are synthesized as pro-proteins, and then cleaved to give the active form, e.g. pro-caspases.

splice variants - alternative splicing may create different sized proteins from the same gene relative charge - the composition of amino acids (charged vs non-charged).

multimers - e.g. dimerisation of a protein. This is usually prevented in reducing conditions, although strong interactions can result in the appearance of higher bands

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.