SuperBlotTM Rapid Single-Step Blocking Solution

(HB9246)
Technical documents: Datasheet
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Product overview

Name SuperBlotTM Rapid Single-Step Blocking Solution
Biological description

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.
  • Ideal for high abundance targets such as loading controls.
  • Works in as little as 15 minutes.
  • Saves hours compared to conventional methods.
  • Acts as a signal enhancer to increase sensitivity.
  • Also suitable for use as a conventional blocking solution.
  • Animal product free.
Description

Rapid single-step blocking solution and signal enhancer for Western Blotting

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Images

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

  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).

Solubility & Handling

Storage instructions

Room temperature

Storage buffer

Contains PBS amongst other constituents

Important This product is for RESEARCH USE ONLY and is not intended for therapeutic or diagnostic use. Not for human or veterinary use

Calculators

Molarity

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Dilution

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More Info

References for SuperBlotTM Rapid Single-Step Blocking Solution

References are publications that support the biological activity of the product
  • An overview of technical considerations for Western blotting applications to physiological research.

    Bass JJ et al (2017) Scandinavian journal of medicine & science in sports 27 : 4-25
  • Western blot: technique, theory, and trouble shooting.

    Mahmood T et al (2012) North American journal of medical sciences 4 : 429-34

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