Can anyone suggest a protocol to verify the oligomerization of secreted protein using immunoprecipitation and native immunoblot.
I am not sure you can use western blotting (if that is what you mean) for looking at oligomerization. Proteins run on SDS/PAGE gels because they are denatured and bind SDS to give them a constant negative charge to mass ration. Sometimes we do this under non-reducing conditions, no DTT or mercaptoethanol to preserve di-sulfide bonds, but the proteins are still denatured and bound to SDS. Under native conditions, such as an IP, proteins are not denatured and will not run on a conventional native acrylamide gel since their charge is likely to be very different, they may even run in the opposite direction. For native proteins, isoelectric focusing is the method of separation, but this does not allow sizing of proteins. Gel filtration is another method and that will give you an estimate of size of a protein complex, but you will not know if the complex is due to oligomerization or if your protein is bound to many different proteins. Oligomerization is not an easy question to address.
Here is how I did the native PAGE to look at purified proteins:
- 2x sample puffer: 3 ml Glycerol; 2,5 ml Tris/HCl (500 mM pH 6,8); 2,3 ml 60% Saccharose; 2,2 ml H2O; Bromophenol blue; don´t heat samples
- running puffer for anode: 25 mM Tris/HCl pH 8,4; 192 mM Glycin
- running puffer for cathode: 25 mM Tris/HCl pH 8,4; 192 mM Glycin; 0,2% Deoxycholate
- prepare gels without SDS (recipe for SDS PAGE with % of acrylamide according to the analyzed protein)
- chill running puffers at 4°C and run gels in the cold room or put the chamber in ice
- run the gel at 45 mA for 30 min before loading the samples
- rinse pockets before loading the samples
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