Goat Anti-Chicken IgY H&L (HRP) ValidAb™

HB9914 shows more intense staining at a given concentration than Thermofisher SA1-300 when used with an anti-GFAP antibody (HB6406) 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 HB6406 at a 1:2,000 dilution. Following washing, the strips were incubated in either HB8356 or Thermofisher SA1-300 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 HB9914. 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 HB6406 at a 1:2,000 dilution or just blocking solution. Following washing, the strips were incubated in HB9914 at either a dilution of 1:10,000 (60ng/ml) or 1:20,000 (30ng/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.

HB9914 is able to produce visible bands when used at dilutions as low as 1:2,000,000 (0.3ng/ml) to stain for GFAP with HB6406 (chicken polyclonal anti-GFAP 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 HB6406 at a 1:2,000 dilution. Following washing, the strips were incubated in HB9914 at either a dilution of 1:500,000 (1.2ng/ml), 1:1,000,000 (0.6ng/ml) or 1:2,000,000 (0.3ng/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.