VMAT2 is a membrane spanning protein. I use SHSY5Y cells and I tried different methods like RIPA buffer, RIPA with 1% Triton-X and in one instance used syringe to lyse the cells. I do not boil my samples (as protein is membrane bound). I load 50 ug protein on 10% Tris-Glycine gel and transfer for 80 min on PVDF in 15% methanol Tris Gylcine buffer. I still do not get a decent signal. Sometimes I see a very light band but not clear at all. If anybody has any suggestions, please share them.
Thanks Amanda,
I did use antibody to detect the signal on PVDF membrane. It is a rabbit polyclonal antibody.
Thanks for all the helpful suggestions.. Some I may not be able to try out as the current funding in the lab is a bit tight.
But I appreciate your reply. Thanks for the link too.
Sorry Vartika, but why don't you warm up your samples in mercaptoethanol? Do you think that you will destroy the epitop with temperature? And if yes why don't you use something milder as dtt?
Skipping protein denaturation (and not destroying the 3D protein structure) will give you a mixture a different protein conformation running at different speed in your gel and resulting to a decrease signal at the predicted molecular weight.
Hi Katerina, Thanks for your suggestion. I havent tried Hepes buffer so I will give that a try next time I run my gel.
Hi Casadei, you make a good point but I experienced that boiling the membrane protein causes it to aggregate due to the hydrophobic patches being exposed. It can be warmed at 50C for 10 mins I suppose. I tried that once with no success. This protein has been notorious for running at a different mol weight than its actual molecular weight due to the membranous nature. It runs at three different molecular weights and also gets gylcosylated.
Hi Vartika,
you are right that for membrane protein boiling causes aggregation. We never heat our SDS-PAGE samples higher then 65°C. Can you express an epitope tagged version of your protein and establish the western using an antibody that worked previously? Could be that a lousy primary antibody causes the problem. In case you are worried that your protein is not solubilized by TX100, would lyze cells directly in SDS sample buffer. Heat the sample for something like 15 min at 65°C. Because of the chomosomal DNA, your sample will be highly viscous. In order to reduce that, you could use a Thermomixer at full speed. Alternatively use a sonicator bath and vortex ... Finally you load the sample on the gel using e.g. a 200 ul Gilson pipet or an Hamilton syringe.
Best, Marius
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