We were planning to site specifically conjugate antibody through strain promoted azide-alkyne cycloaddition (SPAAC) click reaction. In Part A: We initially conjugated Dibenzylcyclooctyne- PEG4-N-Hydroxysuccinimide (DBCO-PEG4-NHS) to antibody by targeting the nucleophilic amines. later the reaction was passed through filtration column (BioSpin) to remove excess DBCO. In part B: We reacted the conjugated antibody with the azide tagged peptide and passed through filtration column to remove excess azide tagged peptide.
Problem1: I was not able to recover any of the antibody tagged peptide in part B, Why?.
Problem 2: The recover during the filtration steps is very low like around (30-40%), why?. - We tried dialysis also as the reaction is in low volume (~50-100ul), it is challenging to recover the mixture from the dialysis cassettes.
what would be the best method to get high recover?.
your suggestions and time would be appreciated
For Part A, what method did you use to characterize your antibody-DBCO conjugate? I would first make sure that the conjugation was successful by MS (ESI, MALDI, etc). If you are sure that DBCO is conjugated to your antibody, then also make sure your reaction conditions are optimized. Typically for DBCO-azide conjugations I allow 16 hours at room temperature for the reaction to go to completion. Finally, your low recovery during filtration could be due to a number of factors, it is hard to determine when using commercial filtration products. You could try switching to regenerated cellulose filters (Amicon Ultra) which I have used to separate biomolecule conjugates from small molecules. Another option is to use a simple gravity flow gel filtration column, that way you can collect fractions and analyze each one to see where your product is coming out. Hope that helps!
Thanks Colin for your suggestions, We use UV spectroscopy (Nanodrop - 309nm)to determine, if the Antibody is conjugated with DBCO, as DBCO adsorbs at 309nm, the conjugation was confirmed by the appearance of the peak at 309nm compared to the unconjugated sample.
UV-Vis is a good way to confirm DBCO-NHS conjugation, but only qualitatively. How many lysine residues does the antibody have? Sometimes it is difficult to control the exact number of DBCO groups conjugated to each antibody.
Nagarjun, as for Step1, you need to make sure all excess of DBCO reagent is removed. Try to use second spin column from a different manufacture. I also would react a small aliquot of Cy5-Azide (https://clickchemistrytools.com/product/cy5-azide/) and remove excess of Cy5-azide. By measuring A280/A650 you will find exactly how many REACTIVE DBCO molecules you put on your AB. If you use small columns like Zeba 0.5 mL it will take very little time and materials, but provide exact number of DBCO molecules per AB.
As for second step I would suggest using Agarose-DBCO (https://clickchemistrytools.com/product/dbco-agarose/) to remove all azide containing molecules if simple desalting does not work at all. After cleaning simple pallet agarose, and keep your AB. Peptides sometimes might be hard to remove with simple spin columns.
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