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

HB8356 shows more intense staining at a given concentration than Sigma A6154 when used with an anti-calretinin antibody (HB6494) in a rat brain cytosol preparation. 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 HB6494 at a 1:2,000 dilution. Following washing, the strips were incubated in either HB8356 or Sigma A6154 at dilutions ranging from 1:4,000 to 1:256,000 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. 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 HB9524. 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 HB6494 at a 1:2,000 dilution or just blocking solution. Following washing, the strips were incubated in HB9524 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 Calretinin with HB6494 (rabbit polyclonal anti-calretinin 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 blocked for 2hrs in 5% non-fat dry milk before being incubated overnight at 4°C in HB6494 at a 1:2,000 dilution. Following washing, the strips were incubated in HB9524 at either a dilution of 1:500,000 (704pg/ml), 1:1,000,000 (352pg/ml) or 1:2,000,000 (176pg/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.