Hrp conjugated sheep anti mouse igg

Serum samples collected from each mouse 42 days after the first immunization were used to determine anti-CII IgG antibody titres using ELISA. In brief, 96-well ELISA microtiter plates (Thermo Fisher Scientific, New York, NY) were first coated with 200 μL of type II bovine collagen (Chondrex, Redmond, WA) and kept at 4 °C overnight. Non-specific binding was blocked with PBS/3% bovine serum albumin (Millipore Sigma, St. Louis, MO) at 37 °C for an hour and then washed three times. Test sera were diluted 2500-fold, added to the well and kept at 4 °C overnight. After incubation, plates were washed 5–7 times with PBS/0.2% Tween-20 and incubated with 1:5000 HRP-conjugated sheep anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) at 4 °C overnight. After thorough washing, ABTS colorimetric substrates (eBioscience, San Diego, CA) were added to the plates. Reactions were stopped by adding H₂SO₄, and the absorbance levels were measured at 450 nm with a microplate reader (TECAN, Durham, NC).

Cells were scraped and lysed in Laemmli sample buffer (2% SDS, 10% Glycerol and 60mM Tris pH 6.8), heated to 95 °C for 5 min and protein concentration was measured using the Lowry protein assay [35 (link)]. Size separation of proteins was achieved on a 3–8% Tris-Acetate gel (Novex, Thermo Fisher Scientific) and proteins were transferred to a PVDF membrane by wet blotting at 300 mA for two hours at 4 °C in transfer buffer (0.4 M Glycine, 5 mM Tris, 20% Methanol). Next, the membrane was blocked in 3% dry skimmed milk in PBS with 0.05% Tween-20 for at least one hour. Primary antibodies against BRCA2 (1:1000, OP95, Calbiochem, San Diego, CA, USA), BRCA1 (1:500, OP92, Merck Millipore), PARP1 (1:5,000, C2-10, Enzo Life Sciences, Farmingdale, NY, USA) and α-tubulin (1:10,000, T5168, Sigma-Aldrich, Saint Louis, MO, USA) were diluted in blocking buffer and incubated overnight at 4 °C. The secondary antibody, HRP-conjugated sheep anti-mouse IgG (1:2000, Jackson ImmunoResearch, Suffolk, UK), was incubated for 2 h at room temperature. Then, ECL substrate was added and the signal was detected on the Alliance 4.7 (Uvitec Cambridge Cleaver Scientific, Warwickshire, UK). The bands were quantified using the ‘Analyze Gels’ function in Image J (version 1.50i) and equal protein loading was verified by the α-tubulin loading control.