I am trying to purify an antibody by affinity chromatography on the immobilized antigen, a 1300Da synthetic peptide, bound via its N-terminus on NHS activated sepharose (the only amino group in the sequence, I also have added a stuffer sequence between the actually desired peptide and the N-terminus). The resin has a theoretical capacity of 20umol/ml, the peptide is 4.5umol/ml (5mg/ml), the column volume is 1ml. Elution is, as "usual", a gradient towards acidic pH. The resin is based on Sepharose CL4B.
Surprisingly, I get very low yield (roughly 0.1% of total antibody), but when I load the flow through a second time on the column, I can elute an equal amount of bound antibody as in the first run.
An ELISA of the fractions on the coated whole protein (the immunogenic sequence has been taken from) reveals that the vast majority of the binding antibody is in the flow through.
When working with immobilized proteins and otherwise the same method, the usual yield is 50 times as much specific antibody.
My conclusion is that the antigen seems to be inaccessible to the antibody. Could it be that most of the antigen is buried in some small pockets of the resin which are not accessible to the antibodies, due to a size exclusion limit, i.e. the antigen is "too small"? The manual of the resin does not say anything in this respect, this is my first affinity purification using an immobilized peptide.
Any ideas?
I think that your antigen did not bound to the NHS sepharose. Does your peptide contain aromatic amino acids to check the absorption after coupling
What was the antigen for making the antibody? The peptide or the whole protein? If the antibody was derived from the whole protein there is a chance that it might just not be binding strongly to your peptide.
Otherwise I agree with Meir above that your peptide probably just did not bind or that only a small amount of it bound to the column. Not aware of any size exclusion limit for NHS sepharose.
The most probably is inadequate binding of the peptide to the column, but there is also another possibility. Your synthetic peptide might be much worse epitope that the same fragment on the protein. Usually conformational epitopes are more common than linear. Synthetic peptide can present different folding/conformation than the same sequence in the protein. You should investigate the coupling of the peptide to the resin and compare reactivity of the peptide with Ab versus protein. Elisa with peptide coating would be good but competitive ELISA (peptide used as inhibitor, wells coated with the protein ) will give more precise results.
(How) Did you check your coupling efficiency ?
Just offering 4.5 µmol peptide does not mean, all of it it has bound.
I agree with Meir Wilchek in that point.
On the other hand, small ligands (like peptides) tend to go deeper into the pores of the agarose than large ones (like proteins), and thus have a higher percentage of ligands unaccessible for large proteins (like antibodies). If you are limited with peptide you might think about coupling it to a carrier (e.g. BSA) and couple that to the column.
Let me help you. I think that you must to maximize the interaction between antibody and pre-fixed antigen (Sepharose beads). So, I suggest you to develop this reaction (equivalent quantities) in a little conical tube with rotational moving overnight, at low temperature. After we submit the mixture to centrifugation and quantify the supernatant using 280 nm absorption and the extinction coefficient of IG. Obviously the concentration of initial antibody was evaluated by the same methodology (absorbance at 280 nm). The difference between concentrations says you about the affinity. If you want or to need BCA (bicinchoninic) protein methodology can be used. Another question that may occur is concerned to the complexity of the crude preparation that contains the target-protein. It must be decreased using methods of enrichment of proteins as salt precipitation before analytical or semi-preparative affinity chromatography.
Thanks for all the suggestions. Here is some additional information:
Currently, I favor the Peptide sequence (also used for immunization): CGSSGxxxxxxG with xxxxxx taken from the protein that I used for coating in ELISA. No Lysines present. The peptide dissolved quickly in water. Coupling was done at a pH of 8..9, 2hrs at room temp and then o/n in the fridge (usual protocol).
A Tyrosine is present and would allow for monitoring coupling (in the next try). There was no monitoring, however, but the activated resin material has been used successfully before and after for other purposes.
BTW, the underlying agarose has a size exclusion limit in the MDa range, so my original concern should be NOT an issue.
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