SuperBlot^TM Rapid Single-Step Blocking Solution

One-Step Blocking Protocol

1. Add both the primary and secondary antibody to around 20ml of SuperBlot™ Rapid Single-Step Blocking Solution.
   1. We recommend using a secondary dilution of 1:20,000
2. Following transfer, incubate the membrane in the antibody containing blocking solution for at least 15 minutes.
   1. The signal intensity will increase with longer duration incubations. A one hour incubation is around the optimal balance between signal intensity and time saving.
   2. It is not recommended to incubate for longer than 4 hours due to the risk of high background staining.
3. Wash the membrane three times for five minutes with either PBS-T or TBS-T
4. Develop the blot using a ECL substrate kit such as SuperBlot™ ECL Western Blotting Substrate Kit (High sensitivity).

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

1. Following transfer, incubate the membrane in around 20ml of SuperBlot™ Rapid Single-Step Blocking Solution for 1-2 hours.
2. 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.
3. Wash the membrane three times for five minutes with either PBS-T or TBS-T
4. Dilute the secondary antibody into SuperBlot™ Rapid Single-Step Blocking Solution and incubate for 1-2 hours at room temperature.
5. Wash the membrane three times for five minutes with either PBS-T or TBS-T
6. Develop the blot using a ECL substrate kit such as SuperBlot™ ECL Western Blotting Substrate Kit (High sensitivity).

Room temperature

Contains PBS amongst other constituents