I am trying to do Co-IP using Pierce Crosslink Immunoprecipitation Kit. However, something is puzzling me. When I do the elution and subsequently visualize by western blot, I quite often get smears. The smears are all of a familiar pattern with a big smear at ~50 kD, ~25 kD and sometimes at >100 kD (see attachment). The attached file shows a typical elution with input material (very faint band at 50kD) in the first lane; flowthrough in the second, washes in the third and fourth and finally elution in the fifth lane.
Does anyone know why this occurs? My first thought was that is was a bleed-out of antibody from the column to the elution (even though it should be crosslinked to the beads), but as I use polyclonal rabbit IgG on the column and visualize with anti-mouse IgG I would not expect this.
I also did not use this special kit before, but I've done quite a lot Co-IPs before... so at the size of your smears at 25, 50 and 100 kDa and because the smears are so much brighter than the band you actually search for, I would say that you somehow destroyed your antibody into its heavy and light chains. sometimes this happens when you use too harsh elution buffers, so a milder one might help you out here... good luck!
Whilst I have not used the kit in question, could it just be that you have successfully eluted what you are looking for, it is just that relative to your input it is very very concentrated? You could try normalising what you run to the input-if you load 1/10th of your sample as input, and load all your eluate and get the smear, assume that all your ligand is bound to the column and try running 1/10th of you eluate. If you get the same banding pattern at roughly the same intensities, then you have succeeded. I do not know what the 100k plus bands are, but you could just elute a sham column to see if it is a rabbit antibody fragment that is cross-reacting with your anti-mouse secondary.
Hej Daniel,
I have not used this kit but I can see that you have used at least 2x more antibodies try to titrate your antibody to see what concentration is needed to Ip your protein then run WB.
Good Luck
I also did not use this special kit before, but I've done quite a lot Co-IPs before... so at the size of your smears at 25, 50 and 100 kDa and because the smears are so much brighter than the band you actually search for, I would say that you somehow destroyed your antibody into its heavy and light chains. sometimes this happens when you use too harsh elution buffers, so a milder one might help you out here... good luck!
25 and 50 are definitely light and heavy chains from my perspective. Bands can also get a little smeary if the lane is overloaded.
I don't know the kit but smearing is probably from the elution conditions (if it's native elution with Glycine pH=2 some proteins might degrade, might need to test for different elution times and conditions) and clearly the antibody chains come out. To avoid that you probably can cross-link your antibodies to the beads (not sure which ones they use in the kit) or use special secondary antibodies that only recognize native primary antibodies, like the trueblot. In this case you have to be able to fully denature your protein samples at 95C, which some proteins also don't like. I do often IPs for a protein at 60 and the cross-linking solved my problem of seeing the IgG from the IP antibody right on top of my band (since I do IP and western with antibodies from the same species)
In addition to above suggestions, you may run SD-PAGE collecting smearing bands aleast few times and proceed with western blot.
Daniel, When you say "input" material do you mean the protein extract before loading onto the resin, and without any antibody in it? If so then the 2 bands should not be light nd heavy chain cross reacting
Yes, I faced this problem even without kits. Some people said the bands 50 and 25 kDa represent different fragments of antibody light chain. I am not sure what 100kDa is. I tried to use Protein A beads to adsorb out these bands--but my band of interest also became very faint. Titrating antibody levels and using a different (milder?) crosslinking methods may be worth trying. It may also be useful to use a totally different method to confirm interaction between your proteins. Hope this helps!
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