membrane protein in SDS-PAGE ! - (Dec/27/2004 )
I've cloned a membrane protein into a vector with pET30 for expression in E.coli expression system. now I want to check if it's expressed but I have problems loading my whole cell lysates on the sds-page.But I can't find the protein in SDS-PAGE.Perhaps it doesn't get soluble!Can someone help me? maybe there is a special buffer to get membrane proteins in solution?
Thanks in advance!
mmm... i have the same problem( a membrane protein)and searching information about this i find "membrane protein fraction". this is a application note from tecan . in the references they name to Dunn and bradd (methods mol. Biol. 19 203). this paper i think talk about our problem but this paper is offline
but the good thing of this note is that they say the follow:
"Preparation of membrane protein sample 1 mL of crude mitochondria (7.5 mg/mL) were prepared according to [Zischka et al., 2003]. Subsequently they were centrifuged for 30 min (25000 g, 4°C). Then, the pellet was treated with 1 mL of 100 mM Na2CO3 for 30 min on ice. After ultracentrifugation (100000 g, 4°C, 30 min) the pellet was washed with 1 mL of 100 mM Na2CO3 and ultracentrifuged again (100000 g, 4°C, 30 min). Finally, the pellet was lysed with 1 mL of FFE-separation media (see below) and centrifuged once more (25000 g, 18°C, 30 min). The supernatant corresponded to 1 mL of mitochondrial membrane proteins having a concentration of 3.4 mg/mL." (i think that is useful with bacteria too.
and FFE-separation media is this:"The separation media (media inlet 2-5) consisted of 7M urea, 2M thiourea, 7% glycerol, 0.03% HPMC as well as 10 mM DTT and 0.1% Triton X-100". the HPMC is hydroxi Propil metil cellulose, this is used in place of poliacrilamide.
sorry about my english
Thank you for reply!
What I am working is a bacteria(E.coli).Do this way work?Do you have some simple ways because I am doing SDS-PAGE only!
first of all, you may not boil your sample (like most soluble proteins) because membrane proteins often aggregate in SDS, particularly when heated. Just add your sample buffer and let it stand for a few minutes at room temp.
You may further want to dilute your sample in sample buffer. Remember that with concentrated samples of bacterial cells you are trying to solubilize several dozens of mg/ml lipid and protein with only ~20 mg/ml (2%) SDS, but you want to achieve an SDS/protein ratio of 1.4 (membrane proteins normally even a little bit more). The best would be to make serial dilutions and load them all onto a gel. Furthermore since membrane prots are normally more hydrophobic than average they bind more SDS than soluble proteins and run faster. So look for you protein at a lower position than expected. E.g. a 45 kDa prot I´m working on runs at apparent 33 kDa.
Hope that helps, good luck,