4 replies to this topic

[kirikiri]

Hi everyone,

I need some help!

So I'm trying to purify a protein (and I've purified other His-tagged proteins before). The odd thing is that both on Coomassie gels as well as with my His-antibody I detect a very specific, strong band at the expected molecular weight of ~25kDa in total protein extracts as well as pellet and soluble fraction, but as soon as I get to the flow-through, wash and elution fractions, the band shifts up to about 30kDa. All it comes in contact with in the wash buffer is imidazole, NaCl and sodium phosphate (well, and the cobalt-column itself). What is going on? Could imidazole somehow bind to the protein?

Thank you so much,
Kiri

[mdfenko]

buffer components (eg salt) in the sample could cause a shift in apparent mw during sds-page.

[kirikiri]

mdfenko, on Apr 1 2010, 03:27 PM, said:

buffer components (eg salt) in the sample could cause a shift in apparent mw during sds-page.

On a denaturing gel, though? I would expect it on a native one, but this shouldn't be affected, correct?

[mdfenko]

kirikiri, on Apr 1 2010, 08:49 PM, said:

mdfenko, on Apr 1 2010, 03:27 PM, said:

buffer components (eg salt) in the sample could cause a shift in apparent mw during sds-page.

On a denaturing gel, though? I would expect it on a native one, but this shouldn't be affected, correct?

salt can have an effect on the individual lane, even on a denaturing gel, by increasing the local conductivity. on sds-page you can get local hot spots caused by salt in the sample.

also, if you have potassium salts present you will get cation exchange with sds and form insoluble kds.

[ProteinWork]

Only mass spec can confirm that your protein's molecular weight is off. Gels could be misleading.