Smartblock pcr 96

A total of 10 µL of 50 mM aqueous tris(2-carboxyethyl) phosphine hydrochloride (TCEP) was added to the 150 µL 2% FA solution containing the purified antibody. The PCR tubes were then placed on the Eppendorf Thermo Mixer with a SmartBlock PCR 96 at 90 °C and 800 rpm for 5 h. Dithiothreitol (DTT) was also tested as a reducing agent during protein cleavage. In that case, a 50 mM DTT solution was added to the 150 µL of 2% FA solution containing the purified antibody. In addition, cleavage times of one, two, three, four, and sixteen hours were tested.

In a 0.2 mL PCR tube, the volume of the antibody solution (usually around 10 µL), corresponding to 10 µg of the antibody, was mixed with a final TCEP concentration of 0.1 mM and sulfuric acid (5 mM) so that the final volume of the solution was 150 µL. Antibody cleavage was carried out for 30 min at 99 °C and 950 rpm on an Eppendorf Thermo Mixer with a SmartBlock PCR 96. No alkylation step was performed. Afterward, peptides were enriched and washed with Pierce C18 tips (10 µL) according to the manufacturer’s protocol. Peptides were eluted from the peptide tip with 2 µL of 2,5-dihydroxyacetophenone (DHAP) MALDI matrix solution (10 mg/mL, 69.9% purified water, 30% ACN, 0.1% TFA) directly on a spot of the MALDI target. MALDI measurements were performed on a Bruker Autoflex maX in reflector mode. The instrument was calibrated with the Bruker Peptide Calibration Standard II using DHAP as the MALDI matrix. A total of 5000 laser shots were accumulated to obtain a fingerprint spectrum.

In a 0.2 mL PCR tube, the volume of the antibody solution (usually around 10 µL) corresponding to 10 µg of the antibody was mixed with a final TCEP concentration of 0.1 mM and 0.1 M Tris buffer (pH 7.8) so that the final volume of the solution was 50 µL. Tris buffer (pH 7.8) was prepared in the following way: 0.7877 g tris(hydroxymethyl)aminomethane and 2.127 g tris(hydroxymethyl)aminomethane hydrochloride were dissolved in 200 mL purified water, and the pH was adjusted to 7.8 by adding a few drops of 1 M HCl if necessary. Denaturation of the antibody and disulfide cleavage was carried out by incubating the solution for 15 min at 99 °C and 950 rpm on an Eppendorf Thermo Mixer with a SmartBlock PCR 96. No alkylation step was performed. Afterward, the solution was cooled down to 55 °C, and trypsin was added in a trypsin-to-antibody mass ratio of 1:120. The digestion was allowed to proceed for another 15 min at 55 °C before 10 µL of a 0.1% TFA solution was added. Peptide purification and MALDI measurements were performed as described in Section 2.3.