I am wondering about your blotting conditions in Western Blot (wet system). I am using the bio-rad system. Running my SDS-page of 30mA for about 1 hour and then blot the proteins on nitrocellulose membrane over night at 4°C at 35V. Next day I boost for 30min at 45V.
After blotting I stain my gels and there is always a lot of protein still on the gel! Sometimes more, sometimes less.
How are your gels looking like?
I load about 30µg. I use a Transfer Buffer (20% 10x TB, 10% Methanol).
Thanks for all recommendations!
Thanks a lot for your answers!
I also assume that my transfer is not complete because once I loaded 100 µg just as a test and on first blot, I had "normal" results on my membrane, while coomassie stain showed a totally blue gel.
Interesstingly I saw an increase/decrease, of proteins of the size of interest, which I wish to see on my membrane on the gel!!! So I just blotted again (this time 120V for 60min) and I got "white" areas, where my protein of interest should be, these white areas were surrounded by a thin black line. I looked this already up in the internet/asked colleges. Seems like "ghost band", so protein overloading...
Try to repeat this with a loading of about 30µg did not bring any benefit.
My protein of interests are below the 50kDa - that's why I run very low volt...
I tryed different transfer buffers, with 10% Methanol and 20% Methanol, with 10% TB and 20% TB...
At the moment I try run the transfer at 35V over night (for about 18h) then boost for 30min at 45V, with 20% TB and 10% Methanol.
dear Alexander
which type of sample you used for protein extraction mean either transgenic plant/animal or any other prokaryotic or eukaryotic systems? because every system have different procedures for protein detection. in my case we used BL21 e.coli expression system. second did you analyze your gel to nitrocellulose with Ponceau staining or not?
regards
Yes Proceau staining I always did, too. There are proteins on the membrane. But it seems like "not the right ones" although I know that it's impossible...
The lysates are from a human cancer cell line.
My last transfer (18h, 35V over night, 45V for 30min @ 4°C) seems to be better than the one (same settings) before... much less proteins left on the gel.
How can the results be so variable?! Possible that the apparatus, the western blot apparatus, have different resistances? Like wire-specific things? And the connectors in the lid are also different, makeing electricity run a bit better/worse?
Dear Alexander
may be your antibodies showed interaction with other cellular proteins. make specific antibodies for the desired protein and then visualized after blotting. i think apparatus has no problem.
Tony Bodell University of Salford
Dear Alexander, yes Ponceau staining is a good idea for initial confirmation that proteins are blotting across but I tend to agree with Saiqa, I don't think it is the equipment. Sometimes the amount of protein transferred to the nitrocellulose substrate can be very little depending on your yield at the expression level and may go undetected by the red stain, specific anti- blotted protein antibody should bind to your target if it is present highlighting your desired protein, again this may be very faint..
Hope this helps, Tony
my opinion, as long as your blot is good, the transfer efficiency is never a problem. i used to check proteins left on gel,and some times very blue, especially the high MW region. Prolonging the time and voltage dose not always help. it might be because of the consumption of the transfering buffer.
I would suggest you first of all to run your gel not above 20 mA, but if you are using 30 mA run your gel in cold room.
I almost get a 95% transfer from SDS gel and 80% in Native gels
Also I would recommend you to transfer you protein for only 1 h at 250 mA/ 100 V
Do not use the transfer buffer for more that 3-4 times because it contains methanol, and each time we use the final concentration of methanol changes, And we must know that methanol facilitates the binding of protein to membrane by removing SDS.
I hope it works for you
Amit
if you protein s there on gel that mens there is no problem in running your gel, the problem is in Transfer. So prepare your transfer buffer 1 liter (14.4gm Glycine, 3 gm tris(mwt: 121) and 10% methanol). Dip your membrane in transfer buffer. Run the transfer at 50V and 400mA for 1.5 hr. And use chilled transfer buffer. After the run is over stain your blot with ponceau to check the presence of band. I hope this will work as i hav transferred bands from range of 10 kDa to 200Kda at this protocol.
good luck
Hey everyone!
Thanks a lot for you ideas!
I will try to work out the problem with every protocol or suggestion you wrote.
Will tell results later.
Thanks for now :)
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