i started with 20 ug of IgG and activated it using the traut's reagent and then performed the column chromatography to remove the traut's reagent. i collected 1 ml aliquots and performed BCA assay to check for my IgG in all the aliquots. the total amount of antibody present in these aliquots (after chromatography) are now amounting to ~600 ug (30 fold)!!!!
Perhaps your column is contaminated with protein from a previous run. BCA is a nonspecific reagent. Please also consider that (according to our experience) Traut's reagent is difficult to control. Hence, we now prefer SIA conjugations for many purposes (see paper).
http://www.mdpi.com/2409-9279/1/1/2
Is it possible that the reduction chemistry interferes with the BCA protein assay as is known for NHS ( https://www.ncbi.nlm.nih.gov/pubmed/21762678 )? You may also wish to quantitate your antibody samples before and after every step using an antibody activity (binding) assay. After all, the proof is in the pudding.
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