Low recoveries after Maxpar labeling
I conjugated one antibody (at the amount of 100ug) per day (to be careful). The recoveries were as below
Antibody A = 0.81mg/mL
Antibody B = 0.65mg/mL
Antibody C = 0.61mg/mL
Antibody D = 0.10mg/mL, *0.12mg/mL
Antibody E = 0.00mg/mL, *0.01mg/mL
*Second trial with 20 minutes partial TCEP reduction, instead of 30 mins.
I read some threads here. And I put more concerns in work:-
1) To solve over reduction: I reduced the length of incubation upon partial TCEP reduction, for I thought the over reduction that causes antibody aggregations.
2) To solve technical problems including pipetting errors: The protocols were followed religiously. At the last step, I resuspended the solution at least ten times, pipetting at least 80% of the volume, being careful not to introduce bubbles. I also wash down the sides of the spin filter.
3) Suspecting the bad lot of spin?: I believe it is unlikely given the satisfying recoveries of antibody A, B and C.
4. All the antibodies are BSA- and gelatin-free.
5. Suspecting the bad lot of antibodies?: I am not sure of how this bad lots could affect the recoveries of antibody D and E. I guess it is all about the stability of antibody? It is unlikely to change the antibody clone for it has the validated specificities (in-house). Would you try some other lot/batch of the same clone? I need to work a bit more to justify the problems.
This post a shot in the dark. And I do hope I can get some constructive feedbacks.