Can I denature the antigen source, such as cell lysate (RIPA), and then use the IB (immunoblotting) antibody as IP (immunoprecipitation) antibody to precipitate target protein from cell lysate?
It depends. Does your "IB" Ab react only with denatured Ag? If it was raised against denatured protein (e.g., a gel slice) that may be the case, but if raised against protein prepared other ways it may react with non-denatured or partially denatured protein. Capture of non-denatured has the advantage of capturing other complexed components. If the Ag must be denatured, I recommend adding 4M urea +1% SDS to your solubilization buffer to denature Ags, then diluting the Ag 1/10 before adding Ab. That reduces both denaturants sufficiently that they do not usually interfere with Ab binding to Ag, or capture of Ab-Ag complexes with Prt G-Sepharose.
Most probably yes. If you are interested in your target protein only (not protein complex) you can even use 2% SDS for quick denaturation and then dilute the lysate 1:20 to have final SDS concentration 0.1% before making IP.
Its depend from used agent (for denaturation), but in most cases - yes
It depends on the antibody quality and the antigen. Try HEPES first and use 5 ug for initial IP. Apply positive control as well. If is not working apply denaturing conditions which usually works better for difficult target proteins. Incubation time with Ab is also important in both cases.
Yes you can, but you want to check out what is the IgG subtype of your Ab. I assume you want to use protein-A or -G for IP. You want to bear in mind that the protein-A and -G have different affinity for different subtypes of IgG from different species. Of course, you can use protein-A/G, which is a fusion protein and show good affinity for all types of IgG. I agree with previous comments, the lysis buffer and later the washing buffer is critical, especially if you want to perform Co-IP.
It depends. Does your "IB" Ab react only with denatured Ag? If it was raised against denatured protein (e.g., a gel slice) that may be the case, but if raised against protein prepared other ways it may react with non-denatured or partially denatured protein. Capture of non-denatured has the advantage of capturing other complexed components. If the Ag must be denatured, I recommend adding 4M urea +1% SDS to your solubilization buffer to denature Ags, then diluting the Ag 1/10 before adding Ab. That reduces both denaturants sufficiently that they do not usually interfere with Ab binding to Ag, or capture of Ab-Ag complexes with Prt G-Sepharose.
As previously described,if your Ab can detect denatured Ag/protein, yes you can. Also, if using RIPA buffer; it needs to be diluted so to reduce the SDS in the final volume to around 0.1%. All the best.
Yes and no, it depends on what kind of antibodies. Some a.b. might work for both IP and Wb while others just for WB. The best thing you can check the product information for antibodies and see what the company recommends.
Yes you can, most antibodies work for both IB and IP but also there are some antibodies not work specially for IP. For denature of the lysates you have to be careful because that might disrupt the protein complex you are looking for.
Yes. I used old cytometry antibodies for immunoprecipitation successfully.
Dear Julia Vainonen
thank you very much for your kind answer.
According to my understanding, I prepare the followed experimental steps, please help to check whether they are correct.
[1] Adding SDS to 2% in to cell lysate,
[2] boil the sample (100 degree for 5 min)
[3] cool the sample
[4] dilute the sample by 1:20 to make the final SDS concentration to be 0.1% before IP.
thank you very much
Dear all
Thank you for your kind answers.
in this experiment, we just hope to precipitate the antigen from the cell lysate by Mab against linear epitope, but I failed to get it. I therefore consider that the linear epitope of target protein might not be exposed well in cell lysate. we therefore hope to denature the cell lysate and then precipitate the target protein. please let me know your protocol if you have direct experience on this.
thank you very much.
xiaoguang
Dear Fernando Arosa
Thank you for your nice suggestion.
According to my understanding, you suggest to use rat anti-protein antibodies, because this will increase the IP efficiency.
I will first order rat anti-IB antibody for this kind of study next, but if rat anti-IB antibody is not available, what others are better among mouse, rabbit and goat anti-IB antibodies?
thank you very much.
xiaoguang
Dear David Allred
Thank you for your clear suggestion and sharing your experience.
My antibody only react with denature protein.
I will try your method "4M urea+ 1% SDS" to denature my sample before IP.
thank you very much.
xiaoguang
Dear Istvan Foldi
Thank you for your nice suggestion.
I am now using protein G, and the present IB antibody is from rabbit. therefore, the binding is strongly. your reminding is quite important for me. I should also consider this point when choosing antibodies.
thanks a lot
Xiaogaung
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