Sds page standard

A total of 15 μl sample was mixed with loading buffer for protein analysis. The sample was then heated to 95 °C for 5 min. The samples and SDS-PAGE standards (Bio-Rad) were loaded into SDS-PAGE gel wells. Electrophoresis was carried out at 160 mA for 1 to 2 h until the standards properly separated. The gel was then removed and put in the appropriate volume Coomassie G-250 stain solution (Bio-Rad), ensuring that the gel was fully covered by staining solution and placed on a horizontal shaker. The staining solution was poured out, and the gel was washed with water for 4 to 24 h. Water was replaced 3 to 5 times during the period until the blue background was almost removed, enhancing the dyeing effect on the protein bands.

The molecular forms of MMP‐8 were detected by a modified enhanced chemiluminescence Western blot analysis kit according to the protocol recommended by the manufacturer (GE Healthcare) and described by Gürsoy et al. (2018 (link)) and Hanemaaijer et al. (1997 ). Briefly, the PISF samples were mixed with Laemmli sample buffer without any reducing reagents and heated for 5 min, followed by protein separation with 11% sodium dodecyl sulfate (SDS)‐polyacrylamide gels with prestained low range molecular weight sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE) standards (Bio‐Rad) as molecular weight marker and human neutrophil (PMN) MMP‐8 (ProteaImmun) as a positive control. Target detections were performed by using a primary antibody, polyclonal anti‐MMP‐8, and a horseradish peroxidase‐linked secondary antibody (GE Healthcare). The immunoblots were quantified by GS‐700 Imaging Densitometer Scanner (Bio‐Rad) using the Quantity One program (Bio‐Rad).

Human plasma containing EDTA from a single healthy individual was obtained from Zen-Bio. One recombinant HRG variant, containing the mutations Pro204 and Asn493, was produced in CHO cell lines by PeproTech. Four other HRG variants, with pairwise combinations of targeted mutations for positions 204 and 493, were produced in HEK293E-253 cells (HEK293) by ImmunoPrecise. HRGs were expressed with sequences matching allelic variants on position 204 or 493 in human population: Pro204/Asn493, Pro204/Ile493, Ser204/Ile493, and Ser204/Asn493.
Cobalt-loaded resin was acquired from Thermo Scientific. Imidazole was purchased from Sigma–Aldrich. Tris–HCl, Tris(2-carboxyethyl)phosphine, chloroacetamide, sodium deoxycholate (SDC), and TFA were purchased from Sigma–Aldrich. Oasis HLB 96-well μElution Plates were purchased from Waters. Sequencing-grade trypsin was obtained from Promega. SialEXO and OpeRATOR were obtained from Genovis. Plasmin was purchased from Abcam. Formic acid (FA) and acetonitrile were acquired from Biosolve Chimie. The SDS-PAGE standards and Imperial Protein Stain were purchased from Bio-Rad and Thermo Scientific, respectively.

Antigens batches (venoms) were analyzed using 10% TRIS-TRICINE gels and stained with Coomassie blue R-250, and molecular weights were estimated using standard broad range markers (unstained SDS-PAGE standards broad range # 1,610,317 Bio-Rad, Hercules, CA, USA). Quantification of volumes and calculation of molecular weights were performed using the software GelAnalyzer 19.1, available at: http://www.gelanalyzer.com/ (accessed on 2 February 2022) [21 ]. Molecular weights were calculated using the known values of the standard broad rank markers (Bio-Rad): 200 kDa, 116 kDa, 97 kDa, 66 kDa, 45 kDa, 31 kDa, 21 kDa, 14 kDa, 6 kDa. To estimate the molecular weight, we used a simple exponential fit approximation of the Rf (retention factor, measured as the band distance migrated/gel length) of each analyzed band. Samples were loaded at a concentration of 1.5 mg/mL and a final volume of 20 µL. Each antibody purification/preparation stage and antivenoms batches were analyzed using 12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) according to Laemmli [22 (link)] and stained with Coomassie blue R-250. Molecular weights were estimated using standard broad range standards (Bio-Rad). Samples were loaded at a concentration of 1.5 mg/mL.