Yes, this is perfectly reasonable.

React the Ab with DBCO-NHS at around pH 8 in an amine-free buffer, and then desalt to give the pure Ab-DBCO derivative. Next, react with your azide-oligo to get the final conjugate. You may or may not need to remove any free oligo, depending on your application. Note: the NHS reaction is fast (

Yes you can react at room temperature, you won't need to stir. However, you have just 5-10ug of Ab? That is not an amount that I would normally think about stirring anyway. What sort of concentration do you have? Normally you would react with Ab at 1-5mg/ml. In principle, you could use a very dilute solution of Ab, but I would be very concerned about the recovery of the derivative after desalting. 5-10ug is a VERY small amount to desalt, you will probably not get a good recovery at all. Quantification of the recovery would be very challenging too. I think you need to use at least 100ug of Ab, preferably at 1mg/ml (i.e. 100ul) and then use a desalting spin column to clean up. Good luck.

Thank you, I would also like to ask if SDS-PAGE to verify if the coupling is successful can separate Ab and Ab-oligo, i.e. show two bands?Or is the SDS-PAGE sensitivity not able to separate them and should I use HPLC analysis?

SDS-PAGE is fine, esp. if you have a long oligo. How many bases do you have? Or what is the molecular weight?

If you have 30 bases or more, do a NON-reducing gel and run conjugate and free antibody (control) on adjacent tracks. You should see Ab at ~150kDa in the control lane. In the conjugate track, ideally, you will not have anything at 150kDa, and only higher molecular weight material. If you do have 150KDa, then some of the antibodies are not labelled and you will need to optimize the reaction further.

You will get MULTIPLE bands above 150kDa if the conjugation has worked (assuming your gel gives you sufficient resolution). I'd use a 4-12%gel. If you do not see discrete bands, you may get a 'smear' instead, but the smear should be higher than the 150 kDa control band. The smear (or discrete bands if you are lucky) comprises Ab+1 oligo, Ab+2, Ab+3 etc. You always get a mixed population with about 5 or so different-sized products. Typically, for efficient optimized conjugation (i.e with no free antibody at the end), most Abs will have 2-4 oligos, with minor quantities of 1 and 5 oligos. There is no point in trying to get 1 oligo per Ab, it simply isn't possible with this type of conjugation. You would need to fractionate post-conjugation if this is important to you.

Finally, the smaller the oligo the harder it is to use non-reducing gel analysis, but it is preferable to use non-reducing gels if you possibly can. If your oligo is small, the band shift on the non-reducing gel is limited and it is harder to see that conjugation has occurred. For small oligos you will have to use reducing gels, with the disadvantage that the H and L chains split, with each potentially appearing as multiple bands (H, H+1, H+2, L, L+1, L+2 etc). So the profiles are quite complex, but if the aim is to see that conjugation has worked at least to some extent, this analysis will tell you. Note that an antibody with two oligos attached, will STILL have 2 unlabelled chains, and an antibody with 1 oligo will have three. Thus, to see 100% band shift of all H and L chains would require a degree of labeling that would completely destroy the antibody.

If your oligo is very small, the L chain +1 and +2 product is the best diagnostic, as the products will appear between L and H on the gel. For slightly larger oligos, you need to look for the H bands shifts. If your oligo is an 'awkward' size, the L+2 can start to appear near the H or H+1.

Hopefully your oligo is fairly big, which makes the analysis simpler (NON-reducing), and SDS gels are great for running multiple trial conjugations in parallel. HPLC analysis is also possible, but obviously it is much slower and requires more expensive equipment.

Thank you again, very attentive answer. Mine is 57 bases, with non-reducing 4-12% gel, the bands are not higher than the control, but I have fluorescence at 150KDa with azide modified fluorescein, and the free fluorescein is under the gel, I am wondering if the sensitivity of the gel is not enough to separate Ab and Ab-oligopoly，Now my strips are a smear

No, I'm almost certain that you have a conjugation issue with the oligo.

57 bases is large, this will give a very significant band shift on a non-reducing gel. Fluorescence detection is very sensitive of course, you may have very little on the 150kDa band. I would suggest you first try to verify the amount of DBCO on the antibody. React Ab-DBCO with fluorescein-azide, then desalt, and then measure absorbance (not fluoresence). This approach can tell you how many DBCO are available per Ab (on average). You really need to have a value of around 3 (2 would be just about acceptable but some Abs won't have any functional groups, so they can never be conjugated).

Is your azide-modified oligo from a commercial supplier? Any mass spec data to prove azide is on most of the oligos? You can check this yourself using DBCO fluorescein or mass spec. DBCO fluorescein has an advantage here in that it will prove if the azide is ACCESSIBLE.

Once you are satisfied that you have active functional groups on both entities, then assuming you have the correct reaction conditions and allow enough time (probably overnight) then the conjugation will proceed very well. You will need an excess of oligo too (i.e. excess over DBCO groups on the Ab).