acrylamide bis crosslinking  (Bio-Rad)

Journal: Toxicon: X

Article Title: Biochemical characterisation and substrate-specific proteolytic diversity of venom metalloproteinases in African puff adders

doi: 10.1016/j.toxcx.2026.100253

Figure Lengend Snippet: SDS-PAGE analysis of the venoms from individual B. arietans snakes A. West and East Africa. The venoms shown, samples from two different snakes from that region, are 1, 2: GHA; 3, 4: KEN; 5, 6: NGA B; 7, 8: TZA. B. Nigeria, Namibia, South Africa and Saudi Arabia. The venoms shown, two samples of each, are 9, 10: Nigeria (NGA A); 11-13: Namibia (NAM 001, 002, 003); 14: South Africa (ZAF); 15: Saudi Arabia (SDA). Both gels are 4-20% acrylamide (BioRad) and were run under reducing conditions. Eight μg of whole venom was loaded per lane and the gel and was stained using Coomassie Blue R250. The molecular weights of the markers (lane M, Thermo Page Ruler for gel A, Promega Broad Range for gel B) are indicated in kDa.

Article Snippet: Samples were prepared for reducing SDS-PAGE analysis by adding sample buffer to a final concentration of 2% SDS, 5% β-mercaptoethanol and then heating at 85 °C for 5 min. Electrophoresis was performed on 4-20% acrylamide gels (BioRad TGX) using a Tris-glycine buffer system, followed by staining with Coomassie Blue R250, colloidal Coomassie (ThermoFisher), silver-staining ( ) or visualisation using a stain-free system (BioRad).

Techniques: SDS Page, Staining

Journal: Toxicon: X

Article Title: Biochemical characterisation and substrate-specific proteolytic diversity of venom metalloproteinases in African puff adders

doi: 10.1016/j.toxcx.2026.100253

Figure Lengend Snippet: Casein degradation assay: Purified SVMPs. The purified SVMPs were incubated with bovine casein, either with or without 5 mM EDTA, for 90 min at 37 C. An aliquot of the reaction mix equivalent to 2 μg of casein was run on SDS-PAGE. The gel used was 4-20% acrylamide (BioRad) and was stained with Coomassie Blue R250. Lane 1, casein control; lanes 2 (no EDTA) and 3 (EDTA) KEN 26 kDa SVMP; lanes 4 (no EDTA) and 5 (EDTA) KEN 68 kDa SVMP; lanes 6 (no EDTA) and 7 (EDTA) NGA 30 kDa SVMP; lanes 8 (no EDTA) and 9 (EDTA) TZA 21 kDa SVMP; lane M, molecular weight markers (Thermo Page Ruler), indicated in kDa.

Article Snippet: Samples were prepared for reducing SDS-PAGE analysis by adding sample buffer to a final concentration of 2% SDS, 5% β-mercaptoethanol and then heating at 85 °C for 5 min. Electrophoresis was performed on 4-20% acrylamide gels (BioRad TGX) using a Tris-glycine buffer system, followed by staining with Coomassie Blue R250, colloidal Coomassie (ThermoFisher), silver-staining ( ) or visualisation using a stain-free system (BioRad).

Techniques: Degradation Assay, Purification, Incubation, SDS Page, Staining, Control, Molecular Weight

Journal: Toxicon: X

Article Title: Biochemical characterisation and substrate-specific proteolytic diversity of venom metalloproteinases in African puff adders

doi: 10.1016/j.toxcx.2026.100253

Figure Lengend Snippet: Effect of non-ionic detergent on the oligomeric structure of the KEN SVMP PIII. SDS-PAGE of KEN SVMP PIII run under various conditions. Lane 1, standard reducing conditions; lane M, molecular weight markers (Thermo PageRuler with key molecular weights indicated in kDa), lane 2 non-reducing conditions (0.5% SDS), lane 3 as lane 2 but with 0.25% NP40 added after the SDS, lane 4, as lane 2 but with 0.25% NP40 added prior to the SDS. The gel used was a 4-20% acrylamide (BioRad) and was stained with Coomassie Blue R250.

Article Snippet: Samples were prepared for reducing SDS-PAGE analysis by adding sample buffer to a final concentration of 2% SDS, 5% β-mercaptoethanol and then heating at 85 °C for 5 min. Electrophoresis was performed on 4-20% acrylamide gels (BioRad TGX) using a Tris-glycine buffer system, followed by staining with Coomassie Blue R250, colloidal Coomassie (ThermoFisher), silver-staining ( ) or visualisation using a stain-free system (BioRad).

Techniques: SDS Page, Molecular Weight, Staining

Journal: Toxicon: X

Article Title: Biochemical characterisation and substrate-specific proteolytic diversity of venom metalloproteinases in African puff adders

doi: 10.1016/j.toxcx.2026.100253

Figure Lengend Snippet: Degradation of fibrinogen, prothrombin and factor X by purified B. arietans SVMP PIs. Fibrinogen (Gel A), prothrombin (Gel B) or factor X (Gel C) were incubated at 37 °C for 120 min with the proteases at a 30:1 (w/w) ratio of substrate:protease. The gel was run under reducing conditions and the equivalent of 1.0 μg (prothrombin, fibrinogen) or 0.4 μg (factor X) of substrate protein was loaded per lane. The gels are 4-20% acrylamide (BioRad) and were stained using Coomassie Blue R250 (prothrombin) or silver stain (fibrinogen and factor X). Lanes: Fb, Pt and FX, fibrinogen, prothrombin and factor X controls (no SVMP); R (gel C only): RVV-X with factor X; lanes GHA, NGA A, ESW and TZA: 21 kDa SVMP PI isolated from venom from these regions; lane KEN 26 kDa SVMP isolated from venom from this region; lanes NGA B and SDA: 30 kDa SVMP isolated from venom from these regions. The molecular weights of key markers (lane M, Promega Broad Range) are indicated in kDa.

Article Snippet: Samples were prepared for reducing SDS-PAGE analysis by adding sample buffer to a final concentration of 2% SDS, 5% β-mercaptoethanol and then heating at 85 °C for 5 min. Electrophoresis was performed on 4-20% acrylamide gels (BioRad TGX) using a Tris-glycine buffer system, followed by staining with Coomassie Blue R250, colloidal Coomassie (ThermoFisher), silver-staining ( ) or visualisation using a stain-free system (BioRad).

Techniques: Purification, Incubation, Staining, Silver Staining, Isolation

Journal: Toxicon: X

Article Title: Biochemical characterisation and substrate-specific proteolytic diversity of venom metalloproteinases in African puff adders

doi: 10.1016/j.toxcx.2026.100253

Figure Lengend Snippet: Degradation of fibrinogen and prothrombin by purified Kenyan B. arietans SVMP PIII. Fibrinogen (Gel A) or prothrombin (Gel B) or factor X (Gel C) were incubated at 37 °C for 120 min with the proteases at a 30:1 or 10:1 (factor X) ratio of substrate:protease. The gel was run under reducing conditions and the equivalent of 1.0 μg of substrate protein (prothrombin, fibrinogen, factor X) was loaded per lane. The gels are 4-20% acrylamide (BioRad) and were stained using Coomassie Blue. Lanes: Fb, Pt and FX: controls, no enzyme; PIII/Fb, PIII/Pt and PIII/FX: enzyme + relevant substrate; PIII: enzyme alone (factor X assay only). The molecular weights of key markers (lane M, Promega Broad Range) are indicated in kDa next to Gel C.

Article Snippet: Samples were prepared for reducing SDS-PAGE analysis by adding sample buffer to a final concentration of 2% SDS, 5% β-mercaptoethanol and then heating at 85 °C for 5 min. Electrophoresis was performed on 4-20% acrylamide gels (BioRad TGX) using a Tris-glycine buffer system, followed by staining with Coomassie Blue R250, colloidal Coomassie (ThermoFisher), silver-staining ( ) or visualisation using a stain-free system (BioRad).

Techniques: Purification, Incubation, Staining

Journal: Toxicon: X

Article Title: Biochemical characterisation and substrate-specific proteolytic diversity of venom metalloproteinases in African puff adders

doi: 10.1016/j.toxcx.2026.100253

Figure Lengend Snippet: Degradation of basement membrane proteins by SVMPs isolated from B. arietans venoms. A basement membrane protein extract (Geltrex) was incubated at 37 °C for 30 min with the purified proteases at a 30:1 (w/w) ratio of substrate:protease. The gel was run under reducing conditions and the equivalent of 8 μg of substrate protein was loaded per lane. The gel is 4-20% acrylamide (BioRad) and was stained using Coomassie Blue R250. Gel A: Activity of the individual SVMPs: Lanes: Gx, controls (no SVMP); lane 1, GHA 21 kDa SVMP PI; lane 2, NGA A 21 kDa SVMP PI; lane 3, ESW 21 kDa SVMP PI; lane 4 TZA 21 kDa SVMP PI; lane 5, KEN 26 kDa SVMP; lane 6, NGA B 30 kDa SVMP PI; lane 7, SDA 30 kDa SVMP. The molecular weights of key markers (lane M, Promega Broad Range) are indicated in kDa. Gel B: Time course of degrading activity of the 21 kDa (TZA) SVMP PI; reaction was stopped after 2-, 5-, 10- and 30-mins incubation; lane 0, basement membrane protein control (= time 0 min).

Article Snippet: Samples were prepared for reducing SDS-PAGE analysis by adding sample buffer to a final concentration of 2% SDS, 5% β-mercaptoethanol and then heating at 85 °C for 5 min. Electrophoresis was performed on 4-20% acrylamide gels (BioRad TGX) using a Tris-glycine buffer system, followed by staining with Coomassie Blue R250, colloidal Coomassie (ThermoFisher), silver-staining ( ) or visualisation using a stain-free system (BioRad).

Techniques: Membrane, Isolation, Incubation, Purification, Staining, Activity Assay, Control

Journal: Toxicon: X

Article Title: Biochemical characterisation and substrate-specific proteolytic diversity of venom metalloproteinases in African puff adders

doi: 10.1016/j.toxcx.2026.100253

Figure Lengend Snippet: Gelatinase activity of native and de-glycosylated KEN SVMP PIII. The zymogram was prepared with 0.2% (w/w) gelatin in a 10% acrylamide SDS-PAGE gel. The gel was run under non-reducing conditions. The zymograms were then incubated as in section and then visualised by staining with Coomassie Blue R250. This zymogram was destained for sufficient time to visualise the Coomassie-stained SVMP PIII bands. Lane 1, untreated SVMP PIII; lane 2 (control) and 3 (+ PNGase F), deglycosylation in the presence of 0.4% NP-40; lane M, molecular weight markers (Thermo PageRuler with molecular weights indicated in kDa).

Article Snippet: Samples were prepared for reducing SDS-PAGE analysis by adding sample buffer to a final concentration of 2% SDS, 5% β-mercaptoethanol and then heating at 85 °C for 5 min. Electrophoresis was performed on 4-20% acrylamide gels (BioRad TGX) using a Tris-glycine buffer system, followed by staining with Coomassie Blue R250, colloidal Coomassie (ThermoFisher), silver-staining ( ) or visualisation using a stain-free system (BioRad).

Techniques: Activity Assay, SDS Page, Incubation, Staining, Control, Molecular Weight