I have tried to purify the IgG from mouse ascitic fluid using Abfrontier Protein G Sepharose fast flow column according to manufacture's protocol. The nanodrop reading is showing concentration of the purified IgG is in the range of 0.90-1.30 mg/mL in 1 mL elution volume. But when I am trying to confirm the same in SDS-PAGE (12%), its showing only a single band of size almost in the middle of 56.2 kDa and 35.8 kDa (Biorad SDS-PAGE broad range marker, unstained). The Ab must be show two bands on gel, one at 50 kDa (Heavy chain) and 25 kDa (Light chain). I have cross checked all buffers, reagents and protocol following in twice and everything was correct. So what would be the possible reasons for getting these kind of results? Any reasons related to the quality of ascitic fluid used?
Kindly enlighten me with your valuable comments and suggestions. Your efforts will be greatly solicited.
The band may not be antibody. You need to run several controls simultaneously to have a proper understanding of the result.
I hope you can resolve the issue. Good luck
@Ramachandramouli Budida
Hi sir,
Thank you for your valuable suggestions.
I didn't run any positive control in the gel. I have added the samples like Input sample, flow though, wash, then elutions. The antibody binding and elution are pH dependent. I am using binding buffer as 1x PBS, 7.4, wash buffer also same, elution buffer as 0.1 M Glycine pH 2.5, elutions neutralised by 1 M Tris HCl, pH 9.0. I read that Ab will bind to the Protein G at higher pH, namely 8-10 pH and dissociation from resin at pH 2.5-2.7. Another important factor is Protein G bind to Fc region of Ab. Then which is that protein ? can you help me in this regard.
Thank you
Shyam
The buffers system looks flawless to me. The higher pH is required for Protein A or Protein L matrices, not for Protein G. Did you see any expected size bands in the input control? I would only go for a purification step if I am sure of antibody presence in the sample. Good luck
@Ramachandramouli Budida
Could you please help me by suggesting the appropriate buffer system for protein G based purification? The protocol which I have used from one of my lab crew member's. He had slightly modified from the manufacture's protocol. He is getting the result also. In my case, I am unable to rule out. I decided to prepare again all the reagents and do once again. Thanks for your suggestions and assistance. I am thankful to you and greatly appreciated as well.
Thanking you
Regards
Shyam
If you have the time you might want to blot the gel and probe with an anti-mouse-HRP antibody. Given that you can see the protein on staining, a quick block, Ab incubation, wash and detect will at least tell you if the protein in question is immunoglobulin. Even better if you can run a bit of commercial mouse Ig as a positive control. Just a thought. Regards. Karl
@K.P. Fischer
Thank you Fischer. I will check it as per your suggestions.
regards
Shyam
It sounds like you have no Ab in the ascites. Rama is correct. Ab should be the predominant species in the crude material on SDS-PAGE (150k non-reducing or 25k & 50k reducing). If you don't see it, then you're wasting effort on purification.
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