I am conjugating Horseradish Peroxidase to antibodies for research, and I have trying to determine how many molecules of sulfo-SMCC are reacting with the primary amines on the Horseradish Peroxidase, before being conjugated to my antibody. So far I have been back calculating after the HRP and antibody conjugation with the use of Ellmans, but this a guess at best. If anyone has any ideas or suggestions I am open.
Assuming you are conjugating the sulfo-SMCC to the amines in the HRP first, you can use something like the kit described at this link
to quantify the number of maleimides on the HRP.
After reacting the sulfo-SMCC with the HRP, run the reaction over a desalting column to get rid of unreacted crosslinker. If you use a spin column like the Zeba series from Pierce (link
, you should get excellent recovery of the HRP with essentially no change in volume; hence, the HRP concentration will be the same as what was loaded onto the column. Take a small bit of the flow-through (i.e. the HRP-SMCC conjugate) and use it for the maleimide assay. You can then get moles sulfo-SMCC/moles HRP.