Capturem Pepsin Miniprep Kit

Capturem Pepsin workflow

Capturem Pepsin workflow. Antibody samples are denatured and reduced before enzymatic digestion. After initial activation of the columns with the included Activation Buffer, the sample solution (50–800 µl) containing the reduced antibody is passed through the spin column via a one-minute centrifugation. Digestion by pepsin occurs on the column, and subsequent peptide fragments are eluted in a second one-minute centrifugation. Eluted peptides are neutralized with the addition of NaOH and ready for downstream analysis.

Digestion of different mouse antibody isotypes with Capturem Pepsin

Digestion of different mouse antibody isotypes with Capturem Pepsin. 100 µg each of mouse IgGs (IgG1, IgG2a, IgG2b, and IgG3) was denatured in TCEP and acetic acid at 75°C for 15 min, and then diluted in 5 formic acid buffer. The diluted samples were digested using Capturem Pepsin spin columns. Both digested and undigested samples (4 µg each) were evaluated by SDS-PAGE.

HPLC profiles of lot-to-lot comparisons of Capturem Pepsin digests

HPLC profiles of lot-to-lot comparisons of Capturem Pepsin digests. 50 µg of Apo was diluted in 5 formic acid and passed through Capturem Pepsin columns from three different lots. HPLC profiles show the same fragments for each sample digestion.

Comparison of digestion with Capturem Pepsin columns versus standard in-solution pepsin methods

Comparison of digestion with Capturem Pepsin columns versus standard in-solution pepsin methods. 50 µg of anti-HER2 mAB was digested using three different methods: Capturem Pepsin, in-solution pepsin incubated for four hours, and in-solution pepsin incubated overnight. MS analysis was performed following digestion. The left shift of the peaks for the in-solution digests and the higher number of peaks overall is indicative of over-digestion. Neither of these results were observed for the Capturem Pepsin digest.