Hi Laura Le Gall . It sounds like you are not fully blocking the PVDF membrane or the secondary is not fully washed away.

I have used 5% BSA, 10% dry milk in TBS with 0.1% Tween-20 to block for 2 hr at RT. Take your time in washing the membrane with TBS 0.1% Tween. Be generous with the washes. Over-washing is never a problem.

Do the same thing with the secondary incubation. Again, be generous with the washes. If your antibodies are good they are not going anywhere.

I agree with Steingrimur. Your problem sounds like a problem with blocking and/or washing the membrane.

I usually block with PBS/0.1% Tween-20/5% dry milk for 2h at room temperature or at 4 °C overnight. Make sure you place your membrane on a shaker at low to moderate speed. Also ensure that you have a sufficient volume of blocking buffer, so that the membrane is fully covered and swimming in blocking buffer. After the blocking step, wash once with just PBS, followed by two 5 min washes with PBS/0.1% Tween-20 (this is to get rid of the dry milk). Then, apply the primary antibody in PBS/0.1% Tween-20/10% FCS (you can include azide, if you want to keep and re-use the antibody). Subsequently, wash three times for 5 min (or longer) with PBS/0.1% Tween-20. Then, apply the secondary antibody in PBS/0.1% Tween-20/5% dry milk (never include azide at this step). And finally, wash again three times for 5 min (or longer) with PBS/0.1% Tween-20. You can even increase the Tween-20 concentration to 0.3% if you fear background. Have one last wash with PBS (without any detergent) before developing the membrane.

Good luck!

Ralf Max Leonhardt added a crucial detail. NEVER allow the membrane to dry at any point even when you are developing it.

Hi Steingrimur Stefansson and Ralf Max Leonhardt !

Many thanks for your answers. That is what I thought too, problems with blocking and/or antibodies washings. Unfortunately all those steps are included in the IBind machine process, I don't control any of the blocking and washings steps. It worked so far but I guess something is going wrong now. I'll try your protocols and hopefully be more lucky. I dried my membranes for storage, but I'll activate them in methanol before starting the new protocol.

I agree with the points made by others. For Western blotting, I follow a similar protocol as described by Ralf. I usually block membranes for 1h at room temperature. When/if required, I include 1-2 washes with PBS/0.3% Tween-20 and this step does help reducing the background.

If your western blots do not work because your membrane turns black quickly. The most logical thing is that you have a blocking problem, as Steingrimur points out. As you indicate, you cannot control it and we must assume there is not the problem. There are two more options: 1. a problem with your reagents, for which I recommend testing them in a dot blot, placing conjugator (1 uL in the established dilution) in the PVDF membrane as antigen, block and then substrate. 2. The other rare possibility is your antigen has endogenous peroxidase, which would damage your WB, turning the entire membrane in black in the presence of the substrate. You can test this, also in a dot blot, by placing your antigen on the membrane and then adding the substrate without the participation of the conjugate or primary antibody, just adding substrate.

I really hope you find a quick answer to your problem

Armando