Immunoprecipitation Problems - (Aug/14/2008 )
I am new to the IP world. I am trying to IP Protein A ( 25 kDa) and look for its intreraction with protein B ( 32 kDa). After IP and PAGE run when I stain wit ponceau S i see a huge band around 50kDa ( Presumably Ig band) in the unbound fraction ( supernatant after centrifuging the beads post incubation) and a much fainter band in the bound fraction lane. Shouldn't i see the opposite? here's all i could think of as potential issues:
1. Too much Ip primary antibody ( I use rabbit polyclonal).
2. Too little beads ( I use Protein G beads blocked with 1% BSA/ PBS )
3. Inadequate binding between antibody and beads.
Any suggestion and advice is welcome
1. Too much Ip primary antibody ( I use rabbit polyclonal).
2. Too little beads ( I use Protein G beads blocked with 1% BSA/ PBS )
3. Inadequate binding between antibody and beads.
Any suggestion and advice is welcome
too much IP primary!!! ...hmm......it is possibly my problem too. I used 3 ug for 200 ul cell lysate which wasn't even too concentrated. and I got too much light and heavy chains.
This is one of the reasons why I like to bind the antibody to the bead and then add lysate. First, you can wash the beads and are left with only the IgG that bound the beads and second, you can block your beads (bound to antibody) to help reduce non-specific binding. I assume you are adding the antibody to the lysate, letting it rotate and then adding the beads. This works fine but it is best only after fully characterizing your antibody to lysate ratio. Unfortunately I would be willing to bet that much of your protein A is bound to the excess antibody which you see in the unbound fraction thereby making your IP very inefficient. Do you see a strong IP of your protein or do you still detect a fair amount in the unbound? If you prefer the antibody in lysate method, which is fine, I would recommend less antibody and/or more beads. As for the antibody binding the beads, make sure you are using the correct beads (if using protein A or G). There are also some commercially available binding buffers which help a little.
rkay447,......thanks so much, I even sent your instructions to my supervisor....next time I'm going to bind the antibody to the beads first. the only problem is that my protein is almost at the same size of the light chain (25KDa) and it'll be hard to differentiate....I'm going to do my best this time.
what I usually do is that first I lyse the cells with 2% CHAPS, then I normalize the protein concentration by bradford assay to have equal volume of total protein in each microtube, then I add the antibody, rotate overnight at 4 degrees, the next day I add the beads, rotate 2 hours, centrifuge and wash 4 times with the same 2% CHAPS, then add sds sample buffer and heat for 10 min at 95 degrees.
I have run out of CHAPS for the moment, and I have to use CHAPS because that's the only detergent which will save the conformational change of Bax during apoptosis.
please see the attached photo, even my negative control gave me bands.
Just a thought...but you could crosslink your pull-down antibody (AB) with DSS (disuccinimidyl suberate) to your bead ( pre-incubation of AB and bead is required). I have found this to work well. The reduction in Heavy and Light chain is significant on the western blot. The protocol is described in "Use of Immunomatrix methods to improve protein-protein interaction detection" by Qoronflech et al., 2002 in the Journal of Biomedicine and Biotechnology. Essentially, the Fc region is covalently bonded to the Bead with the DSS. After the pull down AB is linked to the bead, 1.) all free AB is washed away, 2.) your lysate is indexed to protein load (so all samples have the same amount of protein loaded), 3.) your lysate is added to the AB-linked beads, 4.) incubate the lysate bead mixture for X amount of time while rotating, 5.) wash beads with lysis buffer, and boil with SDS. The great part about this method is the bead-bound AB that will not even boil off in SDS.
It's hard to make out what your photo is showing. Are the four lanes with signal all IPs? If so, this appears to be your heavy and light chains. Which lane is your input? I assume it is supposed to be the first lanes in the gel but there is no signal. First, you must be able to show input. Second, you might want to try protein A beads rather than protein G. Rabbit antibodies tend to bind protein A better. You may need to do the IP for longer than 2 hours. I've seen major differences in efficiency (and background) with longer incubations. It's a balance you have to find. Some IPs work better at 4 hours or even up to overnight but generally your background goes up as well. I find that a couple washes with a higher salt buffer or higher detergent buffer can fix this though. There are a couple ways you can get around the heavy/light chain issue. As steak mentioned~ you can covalently bind the antibody to the bead. I've never gotten this to work well but others swear by it. I think it depends on the antibody and how much patience you have in optimizing. Otherwise, I use proteinA-HRP as a secondary. Protein A is only supposed to bind antibodies in a native conformation and not the denatured antibody from the IP.
Thanks rkay447,
I'll keep that in mind, I'm doing Imunofluorescent now, when I finish with IF I'll get back to IP.