HI i am having problem that, my protein is pure 100% and it was purified by isoelctric focusing, when i run this protein in SDS PAGE, I am seeing more bands, i dont know whya, can please explain me. I am obtaining this prure protein from the Iso elctric focusing gels

thanks in advacne

Is that possible that the protein you isolated from IE gel is actually a complex? So when you run it on SDS-PAGE, the complex is sepereated by denaturing condition.

you can try running denaturing gel and native gel side by side and comapre them...

HI i am having problem that, my protein is pure 100% and it was purified by isoelctric focusing, when i run this protein in SDS PAGE, I am seeing more bands, i dont know whya, can please explain me. I am obtaining this prure protein from the Iso elctric focusing gels

Hi there,may be a degradation of the product, do a native page.otherwise do 2d electrophorosis it will clearly tells u pure or not. there may be a chance in ief that that will seperate on the basis of charge in 2d both charge and size matters and tells that pure or not.

all the best

When Thomas Cech saw this phenomenon in his RNA preps he was convinced there was contamination. What he thought he was observing was additional bands due to degradation which was directed by an RNAse that somehow got into his preps. It turned out that this RNA he was working with was a ribozyme and was self-cleaving. Could it be that this protein is self-cleaving when it is separated from its friends (binding partners) as a regulatory mechanism?

Hi ,
I got two ways that could help you to determain what is that you are seeing in yur gels. As already was said:
Either degradation, the protein is in complex, or it could be ubiquitinated or under other post nuclear modification.
i would do the following.
First run it on a SDS-PAGE and then transfer it and do a western.
With your candidate primary Ab. If you would see more then one band it meens that you have got degradation or modification. (you need to run the sample after incubation with 10% B-ME.
For modification- if it is ubiquitated protein try anti Ub P4D1 from santa cruz is a very good M monoclonal.
You can try other kind of modifications such as phosporilation etc.
If you see just one band after using the candidate protein Ab you can try other Ab's that could detect proteins in a complex.
Maybe when you cut the dot from the 2D you took more then one protein.
What IPG strip u are using 3-10 or narrow?
Hope I helped you a bit.
Ask more question if it didnt help you.
Guy

From my expireance if you try to purify a protein by 2DPAGE you shoud run it in a narow range IPG-strip (or what ever other IEF u are using). Then sned it to mass spec to see if it realy contains just one protein. After clarifing all of the above you can be sure that you are running just one protein.
gsovak wrote several ways to short cut the Mass spec procedure.