Need help with WB/IP sensitivity issues - As in way too sensitive! (Nov/15/2006 )

I've been in my new post for about a month now and I'm trying to get an IP going. I am mainly using an IP method proven to work in my old lab but with some modification based on the reagents they have here. There is only one ab for the protein I am trying to IP so I am having to use the same again to WB with. To try and minimise the heavy chain coming up on the blot I am using mouse-anti rabbit peroxidase conjugated antibody from Jackson that is supposed to recognise only light chain. Eventually I want to try and co-IP another protein whose molecular weight is about 50kD so it would run about the same as my heavy chain.

They seem to be set up for super-dooper sensitive western blotting here. They are using PVDF to blot, ECL plus and hyperfilm. Running comperable levels to what I was doing in my old lab, I have the most horrendous Western blot ever. I don't have scanner access or I would post it. But basically there is a lot of background - I can see it glowing in the dark room . The only sample I can see anything reasonable in is the one that I thought I didn't have enough protein for. So obviously I can be running A LOT less protein than I am now. For the stronger samples - the IP input and FT are just a ladder and I can't make out a discreet band at the right size. Is my secondary antibody x-reacting? (I used it at 1:2000 and primary at 1:10000 as has been previously used). The lanes for IP for both samples are just black and/or quite fuzzy. The light chain is enormous, I could see that on my blot w/o even being in the dark room. Heavy chain seems to come up quite a bit as well although not nearly as strongly.

So I can obviously run less protein and hopefully that would help a bit. I'm not sure what else to try, there seems to be so many problems. Reduce the levels of secondary antibody? I am so used to having lovely clear blots that I don't know where to start! I'm also used to using an automatic processor but here they do it manually (!) so that is taking some getting used to and hopefully my technique will improve.

Ideas greatfully receieved.

I use the regular Jackson secondary (H+L chains), and I go up to 20,000 dilution. I think 2,000 is WAY too low of a dilution. The end concentration comes out to be 40 ng/ml after I dilute.

to avoid to see the Ig on your blot you could :
- biotinylate the primary antibody for the detection, and use streptavidin-HRP
- or you could detect the primary antibody with protein A- HRP, which will detect mostly the native Ig used for the detection, and far less the denatured Ab used for the IP.

Thanks! I don't know where I got 1:2,000 from because I've re-read my product sheet and can see that is WAY too much. No idea what happened but I'm re-doing the blot with the appropriate dilution of secondary and see what happens.