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.

i also think you have no antibody present. You did not say how much ascities you loaded, but recovery of only ~1 mg is very low for ascities.

You say you also ran input sample, flow through, and washes - did you see any antibody bands there? As John said, Ab bands should be predominant in the input and absent in the flow through.

If you are not seeing these bands, perhaps the ascities was not stored properly, and the Ab has degraded, with only a portion of the heavy chain binding. If you do see the bands, then you are not getting binding. Protein G likes neutral or slightly acidic conditions for binding to Ab. Use 20mM phosphate buffer, no salt, pH 6.8-7 to dilute the ascities, check the final pH and titer if necessary to that pH, and filter. Then use that buffer to load. If Ab is present, you might get better binding.

Other suggestions - use 100 mM glycine, pH 2.7 for elution, and neutralize with 1M Tris base (not HCl), at pH 9. Make sure the elution buffer pH is not too low - pH