Peroxidase affinipure goat anti mouse igg h l

Proteins were resolved in 12% SDS-PAGE, transferred to PVDF membranes (GE), and incubated with primary antibodies against HA-Tag (C29F4) (3724, CST, USA), GAPDH (30201ES20, Yeasen, China), mCherry (ab125096, Abcam, UK), α-Tublin (11224-1-AP, Proteintech, USA), his (30404ES60, Yeasen, China). Second antibodies are peroxidase-Conjugated Goat Anti-Rabbit IgG (H+L) (33101ES60, Yeasen, China) and Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (33201ES60, Yeasen, China).

Protein was extracted from cells, liver, and kidney using RIPA lysis buffer (Yeasen). Protein extracts were quantified by the BCA Protein Quantification Kit (Yeasen). For γH2AX, 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to separate the protein bands, and then, proteins were transferred from gel to the PVDF membrane, blocked with 5% milk powder in TBST for 1 hour. Membranes were incubated with specific antibodies for γH2AX (ab81299, Abcam) and tubulin (ab6160, Abcam). For collagen, 8% SDS-PAGE was run. Membranes were incubated with specific antibodies for GAPDH (60004-1-lg, Proteintech) and Collagen I (ab260043, Abcam). Then, immunocomplexes were detected by Peroxidase-Conjugated Goat Anti-Rabbit IgG (H+L) (33101ES60, Yeasen), Peroxidase-Conjugated Goat Anti-Rat IgG (H+L) (33301ES60), and Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) (33201ES60, Yeasen) followed by enhanced chemoluminescence (New Cell & Molecular Biotech). The membrane was finally photographed by imaging techniques (Tanon-4600SF). ImageJ was used to quantify the intensity.