If I need to label an antibody with SMCC-activated R-phycoerythrinand then use the antibody coupled with phycoerythrin as an ELISA, what other reagents are needed? Is there a specific procedure?

If I need to label an antibody with SMCC-activated R-phycoerythrinand then use the antibody coupled with phycoerythrin as an ELISA, what other reagents are needed? Is there a specific procedure?

The main chemicals used are 0.1M PBS, disodium EDTA, NEM, and the reducing agents DTT or 2-MEA, or TCEP, and 10kD ultrafiltration tubes or desalting columns.

Depending on the characteristics of the antibody, it is assumed that the antibody is a conventional hybridoma monoclonal antibody (e.g., mouse IgG 2a ,150kDa), and that the antibody is reduced in a neutral buffer (e.g., PBS+ EDTA,PH7.2) using the appropriate amount of DTT or 2-MEA, exposing the -SH (sulfhydryl group), and then the SMCC-activated dye is added to the reduced antibody (according to a certain ratio) at 37°C or Then add the SMCC-activated dye into the reduced antibody (according to a certain proportion), mix the reaction at 37℃ or 4℃ overnight, and then add chemical kits, such as NEM (N-Ethylmaleimide) to block the reaction for about 0.5 h, that is to say, the labeling is completed; the subsequent can be determined according to the actual situation to carry out further purification operations.

Alternatively, the antibody can be treated with SATA, or 2-iminothiocyanine hydrochloride, and then add the relevant reagents (SATA requires hydroxylamine hydrochloride, etc.) to activate -SH (sulfhydryl), and then add the SMCC-activated dye to the activated antibody (according to a certain ratio), and then mix the reaction overnight at 37℃ or 4℃, and then add the chemical kit, e.g., NEM (N-Ethylmaleimide), to complete the labeling. Ethylmaleimide (NEM) blocking reaction is also possible, this way will not reduce the antibody, but may interfere with the antigen-antibody binding site, the steps are also more complicated.

The choice of which method to use depends on the type and characteristics of the antibody. The proportion of fluorescent dyes to be used also needs to be set up for pre-testing to find out.

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