If the antibody had high affinity for the peptide, it would be very difficult to elute from the column. It might still be there. Try taking a small portion of the resin and boiling it in SDS-PAGE sample buffer, then running the supernatant it on a gel.

Dear Adam and Stjepan, thank you both. Good idea to make SDS-PAGE. To clarify. I eluted the column with 0.1 M glycine and dropped 1 ml of the eluate in 1ml (10 fractions) in 0.3 M phosphate buffer pH 8.0 to neutralize the eluate immediately and measured them by UV 280.

I remember there are also other elution conditions with SCN- (?) at neutral conditions. Has someone experience with this?

The column material is a sulfolink with an iodacetamide group wihich reacts with cysteine. So the peptide can be attached very specifically on the N-terminus.

Greetings from Tuebingen, Germany

I disagree with my esteemed colleagues. As you suspect, some Ab always will bind too tightly to elute, while weak ones will rinse away during your column wash. Some Ab will irreversibly denature upon elution, and some may adhere to the membrane. But from antiserum (not MAb), you should *always* be able to elute some functional Ab from a good affinity column.

Because of your history of success with CNBr, my guess is that your sulfo-link column coupling failed.

Cysteine is prone to air oxidation, especially at the N-ter. This forms a cystine peptide dimer, which is completely unreactive toward I-Ac. The dimer runs much later on RP-HPLC, so it seemed to be consumed by the reaction.

Try reducing the peptide immediately before the coupling reaction. Most people use DTT, followed by careful desalting (e.g., spin column) to remove excess thiol. Desalting small peptides can be tricky. If you use triphenylphosphine for reduction, desalting before coupling is not necessary ;-)

Be sure to degas all of your buffers via vacuum or sparging, and equilibrate the resin in your degased buffer before the reaction.

Thank you John for the possible reasons. In principle I am very experienced in purification of Abs from peptide columns. The coupling of the peptide was controlled by HPLC and looked quite good. The theoretical advantage of the Suldolink column material is the specific coupling via Cysteine on a iodacetamide-functionalized material. But indeed, I never saw it in other labs. Today I used my old CNBr material and coupled my residual peptide, because this never failed. The coupling was perfect after 30 min.Of course, if the Ab is very sensitive to denaturation by low pH, I will get the same result. I will make small portions of gel in a eppi and try different elution conditions and test it by ELISA. I believe it has to do with the material although the irreversibel adsorption should be minimal because of the lots of proteins in serum.

I will inform you. Thank you all and best greetings from the old unversity town Tuebingen, Germany.

Dear Stjepan,

I like how you fight against the cysteine-iodacetamide coupling and perhaps you are right. However I made many successful maleimide-SH couplings and we use the ICH2CONH2" successful in the peptide mass fingerprint analysis for blocking the SH group. "It could work".

The idea was to have the peptide residue free for the interaction with the Ab. This can of course also be done with biotinylated peptide on a Streptavidin column. but let´s see how the normal Sepharose works. And of course the cysteine is for peptide chemists/biochemists the most worse aa.

thank you all.

Dear Stjepan, there is of course no secret. I will write the conditions this evening after my lab, in the moment I am still at home. I agree and I hope that the functionalisation was not properly successful, although according to HPLC the peptide was not detectable after coupling. I also agrree this must something to do with this rare sulfolink resin from Thermo fisher.

I already coupled the peptide to CNBr activates Sepharose and will cyclize overnight.

Good to know to have such kind and helpful colleagues. Thank you. This antibody is isolated from human serum and is very important for me.

Hubert

Dear Stjepan,

enclosed please find the product information

https://www.thermofisher.com/order/catalog/product/20401#/20401

The buffer ist simple 50 mM Tris / 5mM EDTA pH 8.5 (here Tris is allowed in contrast to CNBr) , after 1 hr addition of 50 mM Cysteine and washing with PBS.

The reason why I used this: many years ago I had a simple decapeptide with 3 lysine residues. So for immization I used a N-terminal Cys-version and coupled it to Maleimid-KLH. The immunzation worked perfect. so for purification I used the same methode with the sulfolink gel and got very good and specific antibodies. Now the gel is about 2 years old, stored at 4oC but it might decomposed. But at that time it was perfect.

Why I was totally confused: after cyclisation overnight, I tested the disappearance of the peptide by HPLC and the disappearance of the antibody by Elisa. Before coupling I had a very strong signal on the elisa against the peptide, after the cyclisation no signal. Where is the Ab? Nevertheless tomorrow I will elute my CNBr-coupled peptid column and will see. It was a SARS-CoV-2 Ab-so really like gold....

Best regards

Hubert

I tried to get your email, but the mail was refused. Can you contact me?

[email protected]