37kb and 7kb protein in western blot - (May/14/2015 )

the amount of protein you're loading should be fine.

detergents disrupt migration of proteins, especially smaller size proteins. also, triton will displace sds.

you should try to completely solubilize the pellet. the smaller sized proteins don't necessarily preferentially solubilize.

one other thing, many protease inhibitors are ploypeptides. some of them may be migrating along with your 7 kDa protein.

Thanks for your reply. SO what is your recommendation of a buffer? Should i decrease the concentration of sds? Or remove triton from the buffer?

you can continue to use the lysis buffer but then you should either precipitate the protein and replace the buffer with one that won't interfere with electrophoresis or dialyze to remove the problematic components.

years ago we worked with a protein which was only soluble in a high salt environment. in order to apply the protein to sds-page we would dialyze the protein against sds sample buffer (without tracking dye and glycerol which were added later). this way we removed the salt while we denatured the protein. you can do something similar to remove the detergents, just make sure the dialysis membrane won't pass the proteins of interest.

Thanks for your advice. I ll try that way.

Omg i cannot believe, i saw the band at 7 kb. The membrane is little darker but i can see the band there. Now i just need to tweak to make it better. And the other challenging thing now is showing both the band in the same gel. 37 kb band is missing i was i did the transfer for 15 mins only at 13v. I will play around with volt to figure out the median transfer time and voltage for both the bands. Thank you all for all the comments and suggestion. I ll keep you posted.

what is the formulation of the transfer buffer?

if it includes sds then you must include methanol. in fact, you should include methanol whenever transferring from sds-page to ensure stripping of the sds from the protein so that it won't interfere with binding to the membrane.

39 mM glycine, 48mM tris base, 0.025% sds, 20 % methanol pH 8.3. I even tried with one without sds. Both gave same result. I have another question. The boiled membrane after transfer showed more clear band than ur boiled one. But the only problem is boiled membrane is darker and the bands are lighter. The un boiled membrane has clear background but faint band. I cannot decide which blot to work on.i was wondering how can i get rid of dark background for the membrane thats boiled for 10 mins. Or maybe increase band intensity for un boiled membrane. Transfer time or voltage is not helping. The band is barely there. I even tried increasing the concentration of protein sample. The lane got too darker. I can only tell that the protein is there at 7 kb but it's very difficult to make it appear as a clear distinct band.

Mdfenko, could you plz send me the protocol of your low mw protein extraction along with the recipe of the extraction buffer and cocktail? I wanna start again. I am afraid what I am seeing is not the right band.

the addition of sds in the transfer buffer is generally to facilitate the transfer of high molecular weight proteins. it is usually not necessary for smaller proteins.

why and in what do you boil your membrane after transfer? i could understand it if you transferred from a non-denaturing gel and wanted to denature the protein with sds after transfer but you are using sds-page.

as for high background, you may not be blocking sufficiently or you are using the wrong blocking agent. also, are you boiling before or after blocking?

i have no protocol to give you. i worked with small proteins and peptides but wasn't extracting them. yours should be fine, just remember to remove the detergents after extraction.

After transfer, i keep the membrane in boiling water for 10 mins. This process is for fixation of small protein, i guess. I do my blocking at 5 % milk in TBST overnight at 4C. So is TCA precipitation enough to take off the detergents?