Goat Anti-Mouse IgG H&L (HRP) preadsorbed ValidAb™

HB8356 shows high signal with low background when used to detect GAPDH in a rat brain cytosol preparation in tandem with HB9177 (mouse monoclonal anti-GAPDH antibody). Method: cytosol fractions were prepared from fresh rat brains following established protocols (Molnar et al., 1993. Neuroscience 53:307-326). Rat cytosol samples were loaded (20µg / lane) onto a 4-20% bis-tris acrylamide gel alongside a protein ladder before being run at 160V for 45 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. Following transfer, the membrane was cut into strips using Ponceau dye to visualise and cut individual lanes. Strips were blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB9177 at a 1:2,000 dilution (0.5µg/ml). Following washing, the strips were incubated in HB8356 at dilutions ranging from 1:10,000 (35ng/ml) to 1:640,000 (0.5ng/ml) for 2 hours. Following subsequent washing, detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 6 min exposure, 700nm channel: 30 sec exposure). For more detail please see our Western blotting protocol. Band intensity was calculated using Image Studio version 5.2.5 (LiCor) and a graph was constructed in GraphPad Prism 9 using a 3-parameter Hill equation curve fit.

No background staining is visible when the primary antibody is omitted from the staining procedure using HB8356. Method: cytosol fractions were prepared from fresh rat brains following established protocols (Molnar et al., 1993. Neuroscience 53:307-326). Rat cytosol samples were loaded (20µg / lane) onto a 12% tris-glycine acrylamide gel alongside a protein ladder before being run at 60V for 30 minutes followed by 140V for 80 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. Following transfer, the membrane was cut into strips using Ponceau dye to visualise and cut individual lanes. Strips were blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in either HB9177 at a 1:2,000 dilution (0.5µg/ml) or just blocking solution. Following washing, the strips were incubated in HB8356 at either a dilution of 1:10,000 (35ng/ml) or 1:20,000 (18ng/ml) for 2 hours. Following subsequent washing, detection was accomplished using Clarity Western ECL substrate (BioRad, 1705061) and a Licor Odyssey Fc imaging system (ECL channel: 5 min exposure, 700nm channel: 30 sec exposure). For more detail please see our Western blotting protocol.

HB8356 is able to produce visible bands when used at dilutions as low as 1:2,000,000 (175pg/ml) to stain for GAPDH with HB9177 (mouse monoclonal anti-GAPDH antibody). Method: cytosol fractions were prepared from fresh rat brains following established protocols (Molnar et al., 1993. Neuroscience 53:307-326). Rat cytosol samples were loaded (20µg / lane) onto a 12% tris-glycine acrylamide gel alongside a protein ladder before being run at 60V for 30 minutes followed by 140V for 80 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. Following transfer the membrane was blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB9177 at a 1:2,000 dilution (0.5µg/ml). Following washing, the strips were incubated in HB8356 at either a dilution of 1:500,000 (700pg/ml), 1:1,000,000 (350pg/ml) or 1:2,000,000 (175pg/ml) for 2 hours. Following subsequent washing, detection was accomplished using SuperBlot High Sensitivity ECL substrate (HB9308) and a Licor Odyssey Fc imaging system (ECL channel: 5 min exposure, 700nm channel: 30 sec exposure). For more detail please see our Western blotting protocol.