I'm trying actually to observe a protein of 350 kDa. In order to see this protein I have first separated my protein on a standard 6% PAGE gel. Then I have tried to transfer the protein on a nitrocellulose membrane using three different conditions:
1) Overnight transfer at 100mA, 4°C
2) Transfer 3h at 70V, 4°C
3) Transfer 1h at 100V, 4°C
In the three cases I have used standard Tris/Glycine buffer with 10% ethanol and 0.1% SDS. After the transfer I always stain my membrane with red ponceau. The result of is that in all cases I can see the ladder on the membrane but absolutely nothing else. I have 7 other samples (HEK cells) but it seems that there is no protein on the membrane. I add that I have already used these samples on a 10% PAGE gel and I have obtained a nice and strong staining after transfer so the samples is not the problem.
1. 100 mA o/n transfer looks to be high comparing to standard procedure. so do you see the pre-stained ladder, or you can stain the ladder but not the samples? it is important question. however, i assume you have pre-stained ladder for blotting. ponceau sometimes is tricky to work on. try a short pre-wash in 0.5% (?) acetic acid to remove residual tris-glycine before staining for proteins.
your protein is quite big, i would leave ethanol out completly from the transfer buffer. and you should use o/n protocol.
last of all, i would go further for immunoblotting. it is much more sensitive and if your ladder is on place your proteins have to be there (if the ladder covers the size of your target). more or less.
Thanks for your reply,
Yes I can see the prestained ladder, meaning that in every case the transfer has worked excepted with the third case in which the higher weight (250 kDa) had not transfered. I'll try your prewash with acetic acid.
As I cannot see any protein on the membrane, I have ever stopped at this point. Next time I'll go on further. What voltage do you suggest for an overnight transfer?
30-40 mA for o/n, chilled, and without ethanol. i strongly suggest to try a prestained ladder with MW ranged in your protein for at least the first few experiments. ladder up to 400 kDa exists. its (partial) transfer signs a proper or an appropriate protocol for your protein, even a 2-3 hours one could be working. good luck! and do not expect total transfer at this range.
After performing a new transfer with a 8% gel this morning, it seems the problem comes from the SDS added to the transfer buffer. I have already performed transfer with 8% gel and everything always worked well but this morning I have tried a transfer of a 8% gel using a Tris/Glycine buffer with 10% ethanol and 0.05% SDS (2h at 300mA). Then I washed my membrane with water and I revealed the protein using the red ponceau. Only one column was poorly visible on the nitrocellulose membrane, and it's the column with a strong protein concentration. All the other samples where not visible but the ladder had well transferred. So the only difference between this western experiment and my previous one is the presence of SDS. So could SDS impairs my transfer???
I will try to use the protocol you have mentioned and give some feedback next week.
Thanks a lot
SDS (in the transfer buffer) can help proteins get out of the gel but it also hinders their ability to bind to the nitrocellulose. There is enough SDS in the gel itself to aid gel escape. We routinely transfer using 0.5X Tris/Glycine buffer with 10% methanol (1 hr, 24V, room temp) and have had no issues with detecting ATM (350 kDa) and TSP-1 (450 kDa).
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