Hello everyone,

I am using GlycoLinkTM Immobilization Kit (Thermo scientific 88941) for immobilizing our antibody. However, while using the kit we faced the following problems:

According to the protocol, we quantified the protein after oxidation (followed by desalting) step. At this point, the absorbance at 280nm (A280) was 1.737, corresponding to 1.24 mg of antibody, which is very low as we started with 3.71 mg. Is it usual to observe such loss after desalting step?

In the next step, we added aniline (0.1 M) to the oxidized sample and saved 100ul from this sample for subsequent determination of coupling efficiency.After coupling, the concentration of non-bound protein (from the flow-through) was compared with the starting sample with the following observations:

Using coupling buffer with 0.1M final concentration aniline as a blank, resulted in negative readings of the flow through (non-bound protein). It is hard to believe that all the protein bound to the column and nothing was left in the flow through [We also read Aniline (0.1M) against coupling buffer and it gave an absorbance at 280 of 2.088].

We also tried BCA method for protein quantification and it also produced negative values for the unbound protein and high value for sample (when only coupling buffer and coupling buffer + aniline were used as blank).

We are aware of the interference of aniline with this method of protein quantification.However, we couldn't find any suggestion in the manual/online to avoid any interference from aniline while quantifying the protein. Are there any suggestions regarding the same.

Many thanks in advance!