Hi,

I was running my western blot, and detected a higher molecular band, and have repeated the results several times.

My supervisor wants me to purify and identify this unknown variant form of protein. How should i go about doing it ?

Should i do a FPLC size exclusion column to isolate this higher molecular weight protein? And if so, how can i make sure its pure? Should i run a silver stain just to see if there is only one band, and is that sufficient to say that its pure ?

Help !!!!!

Hi luke
I understand that you don't know anything about the problem protein. The faster way and if you have a little bit of good luck is to purify directly from gel and send it to maldi toff. If your lab have money for try it, run a gel with as much protein as you can, let run the gel as much as you can (for separated the band from others) cut it (trying to get only the band) and send it to maldi toff identificaton. Ask first to the maldi service about the amount needed and the way for do it, but with a normal coomasie staining band cutted with care for don't get dust or human queratine, it is enougth for sequencing. If you tell them the biological source (e. coli, arabidopsis, etc..) they cans ay you if you have one two and wich protein is. If you have more than one then you cna try to get it by fplc purify (Molecular , size exclusion, affinity, ion exchange etc)

Hey thanks kriodos, will discuss with my supervisor about the maldi toff.

To separate the bands on the gel, do you mean that i should just let the smaller proteins run off the gel, and wait for the larger proteins reach the bottom ? Is it possible two proteins could overlap at one size band, and would maldi toff be able to differentiate between two or more proteins ?

yes . First select the % of acrylamide that gets better results for your protein size (8% gel for 24 – 205 kDa proteins, 10% gel for 14-205 kDa proteins and 12% gel for 14-66 kDa proteins). After run the gel and let it run at least to the 4/5 of the gel size (well more or less). Don't let it go out or be just in the bottom. Any way you can get more than one protein in a band. The MALDI will tell you if you have one or more. The results of the MALDI is a different number of peptide chains. then if your protein is in a data base they can identify your protein/proteins (well more or less)
good Luck

Luke said:

Hey thanks kriodos, will discuss with my supervisor about the maldi toff. To separate the bands on the gel, do you mean that i should just let the smaller proteins run off the gel, and wait for the larger proteins reach the bottom ? Is it possible two proteins could overlap at one size band, and would maldi toff be able to differentiate between two or more proteins ?