Advice with WB please - (Jun/02/2014 )

Hi I'm having trouble with my WB's and would be grateful for any advice-

I'm immunoprecipitating MuSK protein from whole cell lysates (C2C12's grown in 6well plates) in order to study its tyrosine phosphorylation under different experimental settings. The IP part is fine in that I have been able to detect MuSK and phosphorylated MuSK but the WB, previously very clean is now horrible looking.

I run MuSK and Phosphotyrosine (PT) blots in parallel but it's the PT blots that have recently become a problem.

I rightly or wrongly have assumed that if it was something systematic like letting the membrane dry out then the MuSK blot would be affected too but this has continued to look great.  With the PT blot there is a lot of background visible after a couple of seconds of exposure but I don't know why. 

I used to use 1:500 of primary antibody and 1:2000 of the secondary and get good results even after an hour of exposure.

I've tried adjusting the following things- different batches of primary antibody (1:500-1:1600 in 5%BSA overnight vs 1hr RT), different batches of secondary antibody (goat anti mouse HRP 1:5000 in 5%BSA 15 min room temp), different batches of BSA block (5% in 0.1% PBS-T overnight at 4 degrees or 1hr at RT) and superblock, different lots of ECL, different boxes of nitrocellulose membrane, manual versus automated development. I've also spun down all my antibodies prior to use and filtered the BSA given the particulate like staining I've been seeing (attached).  I always perform at least 3x5min washes after the primary and secondary antibodies, and try to allow as much excess ECL to drip off the membrane prior to development.  I also tried running the WB in another lab just in case it was something to do with our equipment but this didn't make a difference.

Grateful for any suggestions! 

Is that your molecular weight marker/ladder on the left (dark line)? Looks like you've loaded too much of that. 

How long was the ECL reaction?

And what was your lysis buffer composition? You need phosphatase inhibitors in it for phospho proteins.

This has happened to me before. I tried everything to fix it. Turns out, the company I bought the primary antibody off changed the lot number they were using. I never got a good western with the new lot. Check to see if the company changed the lot number when you bought the primary last. They will often send a different lot number for you to try

Other things that might help is 1.) Additional washes after primary antibody (I will sometimes do six or seven if the antibody is dirty)   2.)Increase the amount of tween in the wash buffer.  3.) Completely drying off almost all ECL

When this happens to me, the thing that actually works best is when I cover up the rest of the blot to leave only your protein of interest showing (or cut out just the area where your protein of interest is rom the membrane and just exposing that).

Hope this helps :-)

Thanks for your advice and comments!

You're right that is my ladder, I've been using a pre-stained Hi Mark ladder.  I usually load 10ul because I find it can be faint despite a good transfer.  I did try loading just 5ul to get rid of that staining but it has still been coming up, not sure why.  

I usually apply the ECL and head straight to the dark room, so probably a minute at the most.

I've been using RIPA buffer from Sigma (150 mM NaCl, 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH 8.0) supplemented with 1:100 protease inhibitor cocktail and HALT phosphatase inhibitors.  I try and do everything at 4 degrees C too, in order to prevent degradation.

Thanks for the tip, I'll give Millipore a call and enquire about the antibody product code.  I'll also increase the wash frequency, tween and try and make sure I get rid of the ECL.

Failing that I like the sound of just exposing the relevant bit.

Thanks v much!