Minimum antibody conjugation

[ssivajothi]

Hi all,

Apologize if this has been addressed at some point.

I would like to know what is the *minimum* amount of antibody that people have successfully conjugated. I know recommended starting point is 100ug. Has anyone done 75ug? 50ug? If you start with 50, do you get anything back at all?

Also, what is the maximum I can conjugate with one tube of the supplied MaxPar polymer? 150ug?

Thank you,
Santhosh

[jlederer]

Hi Santhosh,

We do not conjugate anything less than 100 ug. Most suppliers are accurate with the concentration on the product but some are not. We recommend using a nanodrop to validate protein concentrations prior to conjugations.

The times that we tried 50 ug, it never worked. Probably due to loss of sample during washes. The more antibody that you conjugate, the better the yield. I would recommend scaling up MaxPar reagents at least 50% if you want to do 200 ug.

Hope this helps.

Jim

[GregBehbehani]

Hi Santhosh,

We've done 50ug labeling reactions. It works; you just don't get a very good yield.

There seems to be a fixed amount of antibody that sticks to the filter columns (I think this is about 5-10ug), in addition to a small fraction of antibody that passes through the columns. So when we've done 50ug labeling reactions we get back about 30ug. By the time you factor in all of the cost associated with doing the labeling and QC'ing the antibody it doesn't make much sense in most circumstances.

Best of luck,

Greg

[ssivajothi]

Thank you Jim and Greg for the input.

I strive to start with 100ug whenever possible, but sometimes for rarer antigens the perfect antibody is not available. I am happy if I get back ~25ug in the end. So it is encouraging to hear that starting from 50ug is possible.

I am curious as to why both of your experiences seem to be opposite. Any idea why? And is there something to be extra careful about if starting with lesser material?

Thanks again,
Santhosh