I want to conjugate my antibody with Dylight-NHS ester and corboxylated fluorescent PS beads. How do I check whether both Dylight-NHS ester and corboxylated fluorescent PS beads coupled with my antibody?
Conjugating antibodies to DyLight-NHS ester and carboxylated fluorescent Polystyrene beads involves several key steps:
These are two significantly different reactions.
Ab conjugates to NHS-dye via lysine side chains (epsilon amino) on the Ab replacing the NHS group on the dye, forming a covalent bond. So you'll need to make sure your Ab is in an amine-free buffer. Most people like PBS. You can do the buffer exchange (if necessary) via spin column or dialysis. As Kais Khudhair al Hadrawi suggested, it's otherwise a foolproof "roar and pour" reaction. You don't really need to terminate the reaction, as you'll separate unreacted small molecules afterward (as Kais Khudhair al Hadrawi also suggested). To prove the conjugation worked check the Ab for the expected fluorescence signature.
Coupling your Ab to a COOH bead requires two chemical steps, which may be performed simultaneously, although I recommend you separate between steps. Again, your Ab will react via NH2, so make sure you use an amine-free buffer system. To activate the bead COOH you'll use a carbodiimide reagent. I recommend EDAC. Then separate. Then add the Ab and react. Then wash.
Store the beads in the fridge with a preservative, such as NaN3. You can freeze the conjugated Ab.
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