Larger protein size in SDS gel - why is it like tat? (Feb/09/2007 )

Hi I am wondering what are the reasons for having a larger protein size in SDS gel. I ran my protein (His tagged) which is about 22kDA but on the SDS it appeared as 27kDA...there shouldn't be any post-translational modification (expressed in M15 E.coli) so I am quite puzzled of the size increase.

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molecular weight detrmination on SDS PAGE is only precise if comparing several gels and different standard proteins in a Neville plot;

posttranslational modifications in E. coli is reduced, there is also a lack in post-ribosomal processing; may be there are peptides left which are normally abscised in eukaryotes;

one should also think of checking the gene or better the gene integrated in his vector (restriction analysis, or sequencing)

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Maybe also try size exclusion chromatography. not all proteins will run corresponding to their acutal mass. the binding of SDS to protein is approx. 1,4g per 1g protein. but that's not exactly true for every protein.

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Hey, I have the same problem as well. My protein is 23 KDa, but it appears to be like 29KDa... I guess you have to use a more accurate method to determine mass e.g. ESI spray of the whole protein.

Kev.

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so truee!!!
somebody plsss tell my project leader thiss...
my protein 15 kDa, looks like 12-13 on sds-page.. on using precision plus mol wt markerss.. we havent extensively tried otherss.. but they dont match there too.. i can sayy. i have given reasons like:
1. binding of SDS etc.. for my protein differss..
2. hydrodynamic radiuss.. (now, i think that does exist.. even for the denatured protein.. as a denatured protein.. will not a linear animated strand right!?) so its mobility will also depend on the radius.. of the denatured protein which may differ for marker proteinss..

so.. should these be enough justification for my protein showing up at 12-13 instead of 15?? or cud u add more.. and explain. My protein.. is in no way modified.. i m sure of that.

let me knowww... thankuuu!!!

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you can also include incomplete linearization of the protein (makes it look smaller) to your list of excuses.

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You can add the gel system to your list too - the same protein may have slightly different apparent MWs when you change the gel percentage, gradient, buffers, etc.

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Hi
Including His tag it is 22 KDA? or only ur protein 22 kda? my suggestion is use 15% SDS and run the gel.Use the buffers freshly and cross check the pH. Buffers will give the big difference in mobility.

All the best.

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but my gel system is same for the marker as well as my protein...?!!!?? plss explain! u mean the mol wt marker bands do not represent their true MWs?? thankuuuu!

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Since the amino acid composition and secondary structure of your marker proteins is not the same as your protein of interest, it may be affected differently by changes in the gel system. Also, your markers are typically prepared in a different sample buffer (different conditions with respect to SDS, reducing agent, etc) and may be treated differently before loading (heated or not). The marker proteins usually have been tested thoroughly by the manufacturer to be sure that they run pretty reproducibly, and stay denatured. But your protein of interest is going to be unpredictable. There might be a gel system where you can get this protein to run right where you expect it to be relative to the markers - or there might not.

I've had proteins run a bit high or low many, many times. Generally speaking, it's just something you have to accept - once you've proven to yourself by whatever means are available that it IS the right protein. You could spend months tinkering and never get that protein to run exactly at 22 kDa. Apparent MW by electrophoretic mobility is NOT that same thing as calculated MW, and you can't always expect them to match perfectly. It's a bummer, but unfortunately it's true.

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