反应详情

A 10 mM stock solution of the sulfosuccinimidyl 6-[3′-(2-pyridyldithio)-propionamido]hexanoate (Sulfo-LC-SPDP) is prepared by dissolving 1.3 mg Sulfo-LC-SPDP into 2.07 ml de-ionized water. The conjugation reaction is carried out in phosphate buffered saline (PBS) containing 20 mM sodium phosphate buffer, 150 mM NaCl, 1 mM EDTA and 0.02% sodium azide at pH 7.5. One milligram of lyophilized antibody is dissolved in 450 ml PBS, and 50 ml of Sulfo-LC-SPDP stock solution is added to the antibody solution. The mixture is allowed to react at room temperature for 60 minutes. The sample is applied to a 5 ml desalting polyacrylamide column previously equilibrated with 5 bed volumes (25 ml) of PBS. Fractions are eluted using PBS as the elution buffer, and the protein in the fractions is monitored using a COOMASSIE® Protein Assay (Pierce Chemical Co). Typically, 50 ul of the COOMASSIE® reagent is mixed with 50 ul of each fraction in a micro-titer plate. The COOMASSIE® Blue substrate reacts with the protein, producing a blue color, the intensity of which is dependent upon the amount of protein present in the fraction. The fractions which produce the most intense blue color are those containing the majority of the protein eluted. These fractions are pooled together to produce the disulfide form of the final derivatized product. This is typically the form used for contact printing.

1. customThe conjugation reaction
2. customto react at room temperature for 60 minutes
3. washFractions are eluted
4. additionTypically, 50 ul of the COOMASSIE® reagent is mixed with 50 ul of each fraction in a micro-titer plate
5. customThe COOMASSIE® Blue substrate reacts with the protein
6. customproducing a blue color
7. customThe fractions which produce the most intense blue color
8. washeluted