I am using Polyclonal antibody form rabbit for IP and Polyclonal anti GFP and Anti rabbit HRP for western. I have tried in the reverse way also but still i am getting high background of Protein A/G beads.
Also I need suggestion for IP buffer as well. I am using DTT in my IP buffer does it have any effect during my IP procedure.
For IP, you need 2 primary antibodies, one for the IP and one for WB. You could change one of the two antibodies with another species. A second option is to use acidic glycine to remove the link between your protein and IP antibody. Last solution is to crosslink the IP antibody to protein G beads.
And again yes DTT may modify your IP experiment. For a protein composed two or more polypeptides linked with some disulfide bonds, you may modified these links. And second, your antibody will be reduced by DTT and it will decrease its activity.
hello Christian..,
Here is my buffere recipie... I have use this to get my protein from Arabidopsis nuclei prep.
HEPES-KOH, (pH 7.9) - 20 mM
MgCl2 - 2.5 mM
NaCl - 250 mM
Glycerol - 20%
Triton X-100 - 0.2%
EDTA - 0.2 mM
DTT - 1 mM
I'm a bit confused by your experiment, but if the antibody you are using for IP is from rabbit and your detection antibody is anti-rabbit then if any free antibody remaining from the IP experiment is around you are going to have a very high background. You might consider changing the detection types.
For IP, you need 2 primary antibodies, one for the IP and one for WB. You could change one of the two antibodies with another species. A second option is to use acidic glycine to remove the link between your protein and IP antibody. Last solution is to crosslink the IP antibody to protein G beads.
And again yes DTT may modify your IP experiment. For a protein composed two or more polypeptides linked with some disulfide bonds, you may modified these links. And second, your antibody will be reduced by DTT and it will decrease its activity.
In a best case scenario, you need to use antibodies from two different species - one for IP and second one for detection. As mentioned above, using antibodies from the same species will always give you a background. It could be partially reduced by covalen t crosslinking (immobilization) of your IP antibodies on AffiGel-10 (BioRad), CNBr-Sepharose, or NHS-activated Agarose (Pierce), but you need 0.5-1mg of antibodies to do that. That will give you enough beads to run many IPs. Second option, which I used successfully, is to use biotinylated or enzyme-conjugated antibodies for detection.You can than use streptavidin-HRP or colorimetric substrate for detection with minimal background. This way you can even use the same antibodies for IP and detection.1mM DTT in your buffer should not change thigs much, but you may try to omit it in parallel experiment, just in case.
If you use the same antibody for IP and western, you will get background, simply because you detect the unspecific stuff that your antibody bound. If this is not the problem, you probably need to change your washing conditions.
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