Hi everyone!
I am working on a transciption factor but the available commercial antibodies do not work well in ChIPs, so we have decided to generate our own polyclonal antibody. I have cloned the desirable region of our gene (another lab has successfully used this region for producing their antibody, so specificity and antigenicity has been checked for us !!) and I am about to express a recombinant protein of 250 aa in E.coli that will be used to immunize a rabbit. Because we intend to use it in ChIP (and I guess we want the best epitope recognition) is anything to consider prior the protein purification process? I dont know if it is important at all, but is there any reason to produce it under native or denaturing conditions ? Is purification under native conditions better for "folded epitope recognition" or is it better to use denatured peptide which may expose more epitopes? Any additional tips and advice would be really heplfull!
Thanks,
PTZ
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#2
KPDE
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Posted 12 May 2010 - 01:01 PM
Ptz82, on May 12 2010, 07:36 AM, said:
Hi everyone!
I am working on a transciption factor but the available commercial antibodies do not work well in ChIPs, so we have decided to generate our own polyclonal antibody. I have cloned the desirable region of our gene (another lab has successfully used this region for producing their antibody, so specificity and antigenicity has been checked for us !!) and I am about to express a recombinant protein of 250 aa in E.coli that will be used to immunize a rabbit. Because we intend to use it in ChIP (and I guess we want the best epitope recognition) is anything to consider prior the protein purification process? I dont know if it is important at all, but is there any reason to produce it under native or denaturing conditions ? Is purification under native conditions better for "folded epitope recognition" or is it better to use denatured peptide which may expose more epitopes? Any additional tips and advice would be really heplfull!
Thanks,
PTZ
I am working on a transciption factor but the available commercial antibodies do not work well in ChIPs, so we have decided to generate our own polyclonal antibody. I have cloned the desirable region of our gene (another lab has successfully used this region for producing their antibody, so specificity and antigenicity has been checked for us !!) and I am about to express a recombinant protein of 250 aa in E.coli that will be used to immunize a rabbit. Because we intend to use it in ChIP (and I guess we want the best epitope recognition) is anything to consider prior the protein purification process? I dont know if it is important at all, but is there any reason to produce it under native or denaturing conditions ? Is purification under native conditions better for "folded epitope recognition" or is it better to use denatured peptide which may expose more epitopes? Any additional tips and advice would be really heplfull!
Thanks,
PTZ
Just out of curiosity, why are you using a whole protein rather than a peptide?
#3
Ptz82
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Posted 13 May 2010 - 05:58 AM
Just out of curiosity, why are you using a whole protein rather than a peptide?
[/quote]
Hi KPDE ! Thanks for your interest.
I am not using the whole protein. Instead I use 250aa of a protein of 800aa. And I use this region, because another lab has used this exact region successfully for raising an antibody (they have published it and used it in Western blots, IPs and, importantly, in ChIPs but without saying how exactly they have generated it). And correct me if I am wrong, but I think that for raising good polyclonal antibodies, it is often better to use long peptides (more epitopes) than small peptides (usually synthetic). Also, the available commercial antibodies for our factor of interest were raised against synthetic peptides with poor ChIP performance in my hands.
My question though is if the purification under native conditions of the recombinant peptide to be used for antibody generation is preferable when considering using the antibody for ChIP assays, maybe for the higher probality to recognise conformational epitopes?
Thanks in advance for any comments or advice.
PTZ
[/quote]
Hi KPDE ! Thanks for your interest.
I am not using the whole protein. Instead I use 250aa of a protein of 800aa. And I use this region, because another lab has used this exact region successfully for raising an antibody (they have published it and used it in Western blots, IPs and, importantly, in ChIPs but without saying how exactly they have generated it). And correct me if I am wrong, but I think that for raising good polyclonal antibodies, it is often better to use long peptides (more epitopes) than small peptides (usually synthetic). Also, the available commercial antibodies for our factor of interest were raised against synthetic peptides with poor ChIP performance in my hands.
My question though is if the purification under native conditions of the recombinant peptide to be used for antibody generation is preferable when considering using the antibody for ChIP assays, maybe for the higher probality to recognise conformational epitopes?
Thanks in advance for any comments or advice.
PTZ
#4
Froza
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Posted 05 August 2010 - 04:19 AM
If the antibody is going to be used for ChIP, the antigen should be generated under native condition. Because the protein binding the chromatin with its native conformation, the antibody should recognize both the structure and the amino acid seq..
One the other hand, target protein running SDS-PAGE in denaturing condition in common western blotting, so the antigen should be made in denaturing condition.
One the other hand, target protein running SDS-PAGE in denaturing condition in common western blotting, so the antigen should be made in denaturing condition.
