Conjugation of MCA-Gly Peptides to SH-Carrying Proteins

Weigh 4 mg of a monochloroacetylglycyl peptide (MCA-Gly peptide, peptide carrying a MCA-glycyl residue at the N-terminus) into an Eppendorf tube and add the activated KLH or other iminothiolane-activated carrier protein. Shake vigorously at RT for 3 h. Dialyze the reaction mixture twice at RT against PBS for 1 h each. Calculate protein concentration from 235-, 260-, and 280-nm readings (cf. Protocol 1.1.7). 

Filter the conjugate through a 0.22-pm-tip filter and store in a refrigerator until immunization. For boostering, aliquot the sterile filtrate and freeze at -70 °C or mix with glycerol 1 1 and store at -20 °C. 

Important Prepare conjugates with different carrier proteins for immunization and testing and use different coupling reagents, if possible. 

4-(N-Maieimidomethyi)-Cyciohexane-1-Carbonic Acid N-Hydroxysuccinimide Ester (SMCC) 

Dissolve 4 mg of the carrier protein in 400 pi Soln. A. Dissolve about 0.5 mg SMCC (succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carhoxylate, Mr 334.3) in 50 pi DMF and add this solution to the carrier protein. Shake at RT for 1 h, centrifuge and desalt on a Sephadex G-25 column, equilibrated with Soln. B (the activated carrier appears in the void volume). Cool the receiving tube in an ice bath. 

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