Thu Jul 16, 2015 3:00 pm by mleipold
Glycerol, trehalose, and buffer salts do not interfere with the conjugation reaction (antibody disulfides being cleaved by TCEP to generate free Cys thiols, which then react with the maleimide at the terminus of the metal-loaded polymer).
Gelatin does not contain any Cys, therefore will not interfere with the conjugation reaction either. However, it will usually prevent you from getting an accurate Nanodop A280 reading for your protein yield, as it is also a protein.
Any thiol-based reducing agent (DTT, b-mercaptoethanol, etc) will severely reduce the conjugation efficiency. However, it should not affect the Nanodrop reading.
BSA will severely interfere with the conjugation reaction. BSA has 33 Cys: typically, 1 Cys is free, while 32 are involved in forming 16 disulfide bridges. Once that sees TCEP, some or most of them will break, and will generally outcompete your reduced antibody for reaction with the polymer maleimide. Additionally, like gelatin, BSA will interfere with proper Nanodrop protein recovery determination.
There are BSA removal kits on the market (search on Cytoforum for some recommendations, as this has been a topic in the past). However, the best option, if possible, is just not to use BSA-containing antibody stocks.....it's often worth the extra expense for a custom prep without BSA.
Mike