can you write the detailed protocol which you're using? without it, there's no sense in trying to suggest anything. Write the exact protocol and what protein are you trying to transfer (or at least the mw).

Hey Ghina,

did you check the protein concentrations of your samples before running the blot?

Can you be a bit more specific about what you mean by "not getting tranferred properly"?

Best

Sebastian

what's the MW?

if your proteins are quite big, more than 200kDa, I would advise you to run the transfer overnight (wet system)... if not, if they are smaller, maybe there is a problem with your membrane, transfer system, energy supply. By the way, how does your marker look like?=

I agree with previous repliers: more information about your experiment is needed to solve the problem. In a case of improper transfer I first check whether the running system was ok by comassie blue staining of the gel. Also, I stain my membrane with ponseau. Taking into account that the transfer system works properly and that a buffer is OK, the success of transfer can depend on protein MW: while large proteins require prolonged time of transferring, small peptides should be transferred for a very short period of time. Check out what is a size of membrane pores: possibly, you protein/peptide goes through them? You can put two or three sheets of the membrane and to find out whether you protein/peptides passed the first sheet. You can try different types of membrane: PVDF or nitrocellulose. You can add methanol for better capture of the small peptides. ...

Which system that you have been using? It's a dry or semi-dry one?

can you write the detailed protocol which you're using? without it, there's no sense in trying to suggest anything. Write the exact protocol and what protein are you trying to transfer (or at least the mw).

What transfer system are you using?

What is the molecular weight?

An image would be helpful.

First of all you have to use a proper voltage for the transfer. As said by other friends you should run it at correct voltage for correct amount of time. So your protein size is very important. Secondly sometimes if your protein is small and you are running it for even 2-3 min more it may come out of the membrane into your transfer buffer. So you shold check all these parameters while you are doing a western blot.

The proteins transfert from gel to paper because they are SDS-charged. 1) Don't wash the gel in the tranfert buffer, 2) run a 7.5% Acrylamide gel for 200 kDa proteins, 3) wet the gel with 2/3 ml of SDS running buffer before to assemble

the western blot

good luck

The best transfer, even for high MW-proteins can be reached by tank blots overnight at 15 - 18 °C. If you are using diffusion blots (even with salt driven directed blotting) high MW proteins are very slowly. Also in short time electro blots high-MW proteins needs their time.

... be careful with to much SDS on the membrane. SDS as a detergent has a long hydrophobic tail which will bind to the NC-membrane. A last opportunity could be the use of a PVDF membrane (instead of Nitrocellulose) or even a covalent binding membrane.

Before you are going ahead, make sure, that the protein in on the membrane. Use one strip for a gold staining or use Ponceau-S (this is removable). For the immune reaction part, you need to have a good protein renaturation. In most cases skimmed milk (1 - 3% w/v) is sufficient. The worse detection can depends also on the quality (affinity, specificity) of the used antibody. Prolongation of incubation times for the primary antibody (e.i. overnight at 4 °C) could be helpful as well.

One of my labmate had issue to transfer a protein also, after classical troubleshootting such as increasing the Ab concentration, the transfert time on semi-dry system, test in a wet system, ... She finally checked out the pI of the protein and found that the latest was really high (too high compare to the pH of the transfert buffer) so she increased the pH of the Towbin buffer (transfert buffer) and it worked out great. Good luck !

What percentage of methanol v/v are you using in your transfer protocol?

More information about the transfer is needed: What kind of transfer, time, buffer, temperature...? What is wrong exactly: no protein transfer spotty transferring, small proteins missing, large proteins missing, specific proteins missing?

Try staining the membrane after transfer, and staining the gel before and after transfer to get a better idea of what is going wrong. Coomassie, Ponceau S, silver stains, amido black are some good protein stains.

well sorry for being inactive for so long but alas the project's grant was cancelled so all in vain :/

But I would take time to specially thank all of you for such massive support and reciprocation.

More than 20% of methanol in semi dry transfer can reduce the transfer drastically; specially for high mol weight proteins.