did you stain the membrane with Ponceau red? if not, try these to be sure that there are indeed proteins on your membrane. if you see bands, try if you can get bands for actin/tubulin/gapdh or something like this. if this is not working, it is very likely that something with your AB or ECL is wrong. But it would be easiere to analize the problem ith having some more details... buffers, ecl, AB, concentrations, incubation time...

I agree with Dash and for stain protocol using Ponceau S you can see on my post in Molecular Station Protocols

http://www.molecularstation.com/forum/biology-forum/86153-stripping-pvdf-membrane-ponceau.html

Hi,

I assume that you can not detect a specific band with antibody.

Staining membrane with Ponso s will show that in general proteins were transferred. Same with Commassie, you know the protein went out but that is all. What size bands are you looking for? If protein is too small, depending on the pore size of your membrane and transfer conditions, the protein may simply go through the membrane into the blotting paper. Therefore stain the blotting paper if possible ie in semi-dry transfer. If large size, then it is more likely to stay in the gel and you would see it after commassie blue staining.

What type of transfer do you do? Some type of transfers are better for large proteins whereas others types are better for smaller sizes. Make sure you optimise the transfer condition. Also, are you sure that your primary and secondary antibodies are compatible and are at the right concentrations?

It is difficult to give advice without knowing what type of picture you got. Have you tried different amounts of protein and ECL detection. At times one needs to develop the membrane for ie 20min. What protein are you looking for?

Ask if need more advice. Cheers

It depends on the concentration you are adding. If your protein is expressed at low levels like a cell cycle protein or if it has a lower half life you need to go for higher concentration for loading the gel. First try to go with various conc. like 40, 60, 80 and 100 micrograms/microlitre. I hope you might get to see something on the band