How much can I believe protein MW standards? - (Oct/18/2006 )

Previous students have purified a protein from native tissue and expressed it in bacteria. She ran SDS PAGE gels for commassie and western; got an approximate MW as 48 kDa.

Later on, the same protein purified by another student from the same native tissue runs at 55 kDa in western with the same antibody as before. I purified the recommbinant form of the same protein using the same everything for cell growth, and I also got 55 kDa.

My prof's explaination for the difference in MW is that due to the fact that our MW standards are different (hers and ours), the bands may vary from 48 to 55 kDa.

I am so not convienced by this explaination. Is that much of variation possible?

Thank you in advance.

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possibly I suppose

I bet it's more likely that gel conditions were different and hers was maybe a little warped or something?

it's also possible that hers showed some slight degradation

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Are you using gradient or non-gradient gels?

_Hank

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I see +/- 5 kDa shifts frequently, but I have never sat down and tried to figure out why.

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we have all been running in the same way: 11% SDS-PAGE of full size gels, and for western we all use the same antibodies. The Only difference is the molecular weights standard we use.

Now, I am trying to sequencing the plasmid to check the DNA. Also, purify more proteins to run mass spec to check on the molecular weight again for a higher accuracy.

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If one of the standards is colored, and the other isn't, it could easily account for the difference.

Also, on any gel, MW used to be expressed as MWr; that is, molecular weight relative to the standard used, as an acknowledgment that all proteins whose MW adds up to, for example, 55 kDa do not run the same, even in the same gel system. Somehow we've gotten away from that...

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I agree with homebrew,

you can put your two standards on the same gel, they will not give the same weight.
if you take your stained molecular weight and migrate on tris-tricine or tris-glycine, they will not migrate the same way than a protein non stained, with the same molecular weight.

so, if you want to determine the molecular weight, it's better to use non stained molecular weight standard, and keep in mind that it is relative molecular weight.

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following the literature over the years, uncertainties of +/ 10 % in molecular mass in SDS PAGE and standard reference proteins is not rare; multiple performance, using different reference proteins (without pre-staining) may give a good statistical approach

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thanks guys for your suggestions.
I guess my prof may be right. Anyhow, I will figure it out soon.

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Dear kidroc:
1. protein may migrate different in different page system;
2. we are always doing protein marker, normally, the MW of unstained protein marker is right (+/- 1 kDa); but the prestained marker is not right for the dye can affect the migration of protein different. and the different dye affect diffetently; the MW of protein will be added 5kDa to 15kDa;
3. when you treated you sample with loading buffer, you may treated longer, or added with new DTT or TCEP.

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