How to extract Large protein from Low percentage SDS-Glycine-PAGE gel? - (Jul/07/2008 )

Hi guys:

I have some problem to blots large protein (>250kDa) from 7.5% SDS-PAGE onto PVDF membrane for N-terminal sequencing, I have tried both Semi-dry blot and tank blot, which did not work for me (I use prestained marker (covalent bounded to the marker for blotting purpose) to see if high molecular mass bands to blot over), I wasn't successful.

So I thought about following:

1. electroelution, we don't have such apparatus in our department (Recently I found out no one has it in the country .... this is South Africa I am in). So I opion for that is out

2. Altering the precentage ratio acrylamide : bisacrylamide................. doesn't work for me either?

3. Extended blotting time for both semi-dry and tank blot......... doesn't work

4. Redissolve polyacrylamide gel, and extract protein from it.............don't have protocol

5. Tryspin digest the bands and elute from gel.............We have been waiting and waiting is already been 3 month for Trypsin to get in to this country


So any suggestions are greatly appreciated.



Another thing, does anyone knows the institute or company that can perform N-terminal, internal and C-terminal sequencing, and doesn't cost too much. I come from a very poor country and from poor institute




Your assistances is greatly appreciated


-divinelight-

For wet transfer, have you tried reducing methanol concentration and adding a little SDS to the transfer buffer.

I found this which seems to have good tips

http://www.abrf.org/Other/ABRFMeetings/tut...es/5/index.html

Probably in-gel tryptic digest would be the best to identify your protein. I think Sigma sells sequencing grade trypsin - can't get that in fast? Sounds like you probably don't have mass specs to identify the protein. So maybe a company that does mass spec protein ID will also do your in-gel digest if you cut the band and sent it out? No idea about costs, sorry. If you go this route then make sure you use the stain that the company specifies.

Good luck.