SuperBlot™ Rapid Single-Step Blocking Solution is a novel single-step blocking solution for Western Blot that enables the rapid blocking and staining of membranes in a single stage:
Enables blocking, primary and secondary antibody incubation all in one single step.
Figure 1. Speedy GAPDH staining in rat brain samples using SuperBlot™ Rapid Single-Step Blocking Solution
Rat brain cytosol fractions were loaded (see Molnar et al., 1993. Neuroscience 53:307-326) at 20µg / lane onto a 4-20% tris-glycine acrylamide gel alongside a protein ladder before being run at 160V for 60 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was then incubated simultaneously in SuperBlot™ Rapid Single-Step Blocking Solution, HB9177 (mouse monoclonal anti-GAPDH, 1:4,000, 0.25μg/ml) and HB8356 (goat polyclonal anti-mouse HRP, 1:20,000) for 60 minutes. Following washing, the membrane was developed using a ECL substrate kit 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.
Figure 2. SuperBlot™ Rapid Single-Step Blocking Solution enables signal detection in as little as 15 minutes.
Rat brain cytosol fractions were loaded (see Molnar et al., 1993. Neuroscience 53:307-326) at 20µg / lane onto a 4-20% tris-glycine acrylamide gel alongside a protein ladder before being run at 160V for 60 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was then cut into strips and incubated for either 15, 30, 60 or 120 minutes in SuperBlot™ Rapid Single-Step Blocking Solution, HB9177 (mouse monoclonal anti-GAPDH, 1:4,000, 0.25μg/ml) and HB8356 (goat polyclonal anti-mouse HRP, 1:20,000). After the incubation each strip was placed in wash buffer and then subsequently washed together so that they could be imaged at the same time. The strips were developed using a ECL substrate kit 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.
Figure 1. Speedy GAPDH staining in rat brain samples using SuperBlot™ Rapid Single-Step Blocking Solution
Rat brain cytosol fractions were loaded (see Molnar et al., 1993. Neuroscience 53:307-326) at 20µg / lane onto a 4-20% tris-glycine acrylamide gel alongside a protein ladder before being run at 160V for 60 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was then incubated simultaneously in SuperBlot™ Rapid Single-Step Blocking Solution, HB9177 (mouse monoclonal anti-GAPDH, 1:4,000, 0.25μg/ml) and HB8356 (goat polyclonal anti-mouse HRP, 1:20,000) for 60 minutes. Following washing, the membrane was developed using a ECL substrate kit 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.
Figure 2. SuperBlot™ Rapid Single-Step Blocking Solution enables signal detection in as little as 15 minutes.
Rat brain cytosol fractions were loaded (see Molnar et al., 1993. Neuroscience 53:307-326) at 20µg / lane onto a 4-20% tris-glycine acrylamide gel alongside a protein ladder before being run at 160V for 60 minutes. Wet transfer to a PVDF membrane was completed in 90 minutes using 400mA. The membrane was then cut into strips and incubated for either 15, 30, 60 or 120 minutes in SuperBlot™ Rapid Single-Step Blocking Solution, HB9177 (mouse monoclonal anti-GAPDH, 1:4,000, 0.25μg/ml) and HB8356 (goat polyclonal anti-mouse HRP, 1:20,000). After the incubation each strip was placed in wash buffer and then subsequently washed together so that they could be imaged at the same time. The strips were developed using a ECL substrate kit 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.
Biological Data
Application notes
One-Step Blocking Protocol
Add both the primary and secondary antibody to around 20ml of SuperBlot™ Rapid Single-Step Blocking Solution.
We recommend using a secondary dilution of 1:20,000
Following transfer, incubate the membrane in the antibody containing blocking solution for at least 15 minutes.
The signal intensity will increase with longer duration incubations. A one hour incubation is around the optimal balance between signal intensity and time saving.
It is not recommended to incubate for longer than 4 hours due to the risk of high background staining.
Wash the membrane three times for five minutes with either PBS-T or TBS-T
For more information about Western Blotting including buffer recipes please see our Western Blot Protocol. It is possible to store and re-use the mixed antibodies in blocking solution at 4°C for up to a month.
Two-Step Blocking Protocol
Following transfer, incubate the membrane in around 20ml of SuperBlot™ Rapid Single-Step Blocking Solution for 1-2 hours.
Dilute the primary antibody into SuperBlot™ Rapid Single-Step Blocking Solution and incubate for either 1-2 hours at room temperature or overnight at 4°C. To reduce antibody useage it is possible to use as little as 1ml with the membrane placed into a heatsealed plastic bag.
Wash the membrane three times for five minutes with either PBS-T or TBS-T
Dilute the secondary antibody into SuperBlot™ Rapid Single-Step Blocking Solution and incubate for 1-2 hours at room temperature.
Wash the membrane three times for five minutes with either PBS-T or TBS-T