Transfer high molecular weight protein - (Jun/15/2006 )
I tried to transfer my interesting protein which has high MW (~390 KDa) to Nitrocellulose membrane. I used a 4% gel and this transfer buffer:
Tris Glycine 1X
I transfered with 80°C for 2 hours at 4°C
I stained the membrane with Ponceau S, but there was no band at the molecular weight I wanted
thanks in advance
Try to use 10% MetOH instead of 20%. If you lower the concentration, it is better for high MW proteins. And if you still don't see anything with Ponceau S, before you trow away your blot, I would try and stain with Comassie because it's more sensitive. When I'm not sure about the transfer, I also stain the gel (after the transfer) with Comassie, so that I can see which bands I didn't transfer.
try without SDS and do it in semi dry conditions overnight 5V.
i use as raffaela 10%methanol.
my protein is also very large - i run on 3% low melt agarose with a 15% acrylamice plug for about 3 hours at 100V in TBE/01% SDS. I then transfer by capillary action to NC membrane (in the style of a northern/southern transfer) using TBS/0.04% SDS......seems to work well....
You can try a wet blotting transfer overnight.
And for my protein (220 kDa), the Ponceau staining did not give a good result but when I hybridize with the specific antibody I had a specific band on my blot. So I won't trust Ponceau staining for such molecular weight.