Hi, there
I am wondering why the result of intact IgG in the sds-page is ca. 200-250 kDa? This is a Mouse IgG2b, κ. Shouldn't the MW of a mouse be around 160 kDa?
Now I suspect there might be problems in the sample preparation procedure for sds-page, which led to coagulation. Here is the procedure I used in brief:
1. take 4µL(4mg) mAb and add PBS to 30µL, just to keep the same volume of the adjacent wells.
2.add 4X sample buffer on ice.
3. boil w/o reducing agents at 90°C for 5 min
4. place on ice, ready for electrophoresis
I am looking forward to your kindly feedback. Thanks
The mW of an IgG is around 150 kDa.
When running a non-denaturing (without beta-mercaptoethanol) SDS-PAGE the apparent kDa appears higher, because of the glycosylation of the Fc part. In this non-denaturing SDS-PAGE the IgG band should run at around 200-250 kDA (as observed by you). This protein band is also not distinct, the band should be blurry because of the nonuniform glycosylation pattern.
Best,
Michael
Hi, you could to probe add B-mercapto instantly at moment of prepearing your sample, or add 50% plus B-mercapto, in this reducing conditions you must to obtain 3 bands (one very faint at 160kDa, one more prominent at 50 kDa and last one scattered at 25 kDa) and in non-reducing conditions only one band at 160kDa. You can also try to boil by 10 minutes instead of 5.
Thank you for your kindly feedback, Prof Hust. It is fairly reasonable for IgG to have higher MW observed in a non-denaturing PAGE/Native PAGE.
However, the electrophoresis that resulted in higher MW was performed in Non-reducing condition, rather than in non-denaturing.
In short, the method I used was conventional Tris-glycine SDS-PAGE without any reducing agents(eg: 2-ME). To my understanding, the PAGEs with SDS are all categorized into denaturing type because of SDS, and the protein samples are no longer in their native form. This is the reason why I did not expect to see the band higher than 150 kDa.
Thank you for your idea, David Lule. It is a brilliant way to prove the intact IgG MW by "25*2+50*2=150", but I kinda worry it's too "alternative", isn't it?
Caleb Lin: You're partially correct about native vs denaturing PAGE. It's true that the presence or absence of SDS is one factor. But antibodies have multiple disulfide bonds, both within each of the four polypeptide chains, and between them. Without a reducing agent like 2-ME or DTT, the four chains will remain linked together by disulfide bonds, and each chain will not be unfolded to the same degree as they would if reduced. I haven't actually done the experiment, but I would not expect four disulfide-linked chains to have the same electrophoretic mobility as a single chain with the same mass.
In short, it's slightly more complicated than native vs denaturing, and you might get different results from a "native" gel, "native" reducing gel, a SDS non-reducing gel, and a SDS reducing gel.
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