I got a solution that is highly recognized by ELISA, but after HPLC purification (C18 column), none of the elutions are recognized anymore. Is it possible that the Elution Buffer in some way made my protein/peptide not be recognized anymore?
PS1: I tried to use way more antibody to make the ELISA works.
PS2: After elution, i used SpeedVac to dry my sample and it was resuspended in water.
I agree with Kou Hayakawa at that binding proteins generally show multiple affinities to wide range of substrates or antigens.
What is the size of the protein? what was the maximum concentration of organic solvent? After drying, did the material redissolve? Is it a tagged protein?
I dont know the size exactly, it is probably a peptide. The max concentration was 60% but the last elution was at 50%. Its not tagged and all elutions redissolved after dry.
Hi there,
Are you sure your protein is efficiently eluted? It might be that it behaves badly during the HPLC.
If you do not know your protein, how do you anticipate that it is going to survive the hplc separation?
Perhaps you collected an elution that isn't the protein you intended. Try to check with a standard, and also confirm its purity. As stated by Prof. Ortega, try to understand the properties of your protein in detail, so to subject it to conditions that won't lose it.
I agree with Kou Hayakawa at that binding proteins generally show multiple affinities to wide range of substrates or antigens.
I agree with Dr Hayakawa.
By other hand, you also need to know how much protein do you have in the fractions obtained after purification. In ddition, you can also add to a standard in your ELISA, and verify that your technique and your kit reagents are working correctly. Maybe the buffer is not correct (pH, other characteristics) or incompatible with the ELISA kit reagents.
Success and greetings!
I suspect that your antigen was denatured by exposure to RPHPLC organic phase followed by lyophilization. Try ion exchange and/or sizing, instead or reverse phase, and avoid freeze-drying.
If you still have an aliquot of the sample that you loaded on to the column, you could do a protein assay of that aliquot and all the material that eluted from the column. If there is a discrepancy, then it is possible that your protein is still bound to the column. You could elute with a higher concentration of the organic solvent, or repeat the purification with a different chromatographic medium. If you want continue with reverse phase chromatography, you could try a C8 column. The other possibility is that your antibody recognizes a native conformation of the protein, and the protein lost that conformation during the Speedvac process. You could perhaps test this by subjecting a small amount of the sample that you loaded on to the column to the same process and checking it for antibody binding. If you lose antibody binding, then you have an answer to your question. Try a different method of concentrating your protein down. If you don't lose antibody binding, you will not have an answer because sometimes the presence of other proteins might stabilize your protein of interest. That protective effect would not be there if the fraction with your protein is relatively pure.
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