Anti rabbit igg hrp antibody

Tissue and cells were homogenized in 1× RIPA with 1% protease/phosphatase inhibitor, followed by sonication. Samples were prepared with 2× SDS Page buffer (125 mM Tris pH7, 4% SDS, 0.2% bromophenol blue, 20% glycerol, 5% β-mercaptoethanol) and boiled at 98 °C for 5 min prior to loading. Proteins were separated in 4–20% Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-rad (Hercules, CA, USA), #456-8093) and transferred to Trans-Blot Turbo Mini Nitrocellulose Transfer Packs (Bio-rad, #170-4158). Membranes were blocked in 5% nonfat milk (Biorad, #170-6404) diluted in 1× Tris Buffered Saline, 0.1% Tween 20 (TBST) for 1 h and incubated in primary antibodies at 4 °C overnight with rotation. Membranes were rinsed 5 times with TBST and incubated at room temperature for 1 h in secondary antibody. The membranes were then rinsed 5 times in TBST for 10 min each and imaged on a Bio-Rad ChemiDoc MP system with SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher (Waltham, MA, USA), #34096). Primary antibodies were used at 1:1000 and included p-p44/p42 MAPK (t202/y204) (phospho-ERK1/2) (Cell Signaling (Danvers, MA, USA); No. 4270S) and p44/42 MAPK (ERK1/2) (Cell Signaling; No. 4695S. Secondary anti-rabbit IgG HRP antibody (Sigma (Burlington, MA, USA); No: A9169) was used at 1:7500. Ponceau S (Millipore Sigma; No: P7170) was performed as need for protein normalization.

Protein was harvested using 1X RIPA, prepared with 2X SDS Laemmli buffer (125mM Tris pH7, 10% glycerol, 2% SDS, 5% β-mercaptoethanol, and 0.05% bromophenol blue) and separated via Bio-Rad mini blot gel electrophoresis chamber with 4–20% Mini-PROTEAN TGX Stain-Free Protein Gels (Bio-Rad, #456–8093). Protein was transferred to Trans-Blot Turbo Mini Nitrocellulose Transfer Packs (Bio-Rad, #170–4158) with Bio-Rad Trans-Blot Turbo semi-dry transfer system and blocked with 5% dry fat milk diluted in 1X TBS- 1% Tween (TBS-T). Membranes were incubated in primary antibodies diluted in 5% block (1:1000) overnight at 4 °C and included: p-AKT (#4060S), p-ERK1/2 (#4370S), AKT (#2920S) and ERK1/2 (#4695S) (Cell Signaling). Following washes in 1X TBS-T, blots were probed with anti-rabbit IgG HRP antibody (Sigma, #A9169, 1:7500) in 5% block for 1 h at room temperature, washed with 1X TBS-T, and exposed to West Femto maximum sensitivity substrate (ThermoFisher, Waltham, MA, USA, #34096) or Pierce ECL Western blotting substrate (ThermoFisher, #32209) prior to imaging on a Bio-Rad ChemiDoc MP Imaging System. Relative Densities of bands were measured in Fiji, normalized, and compared in GraphPad Prism.