RapidStep™ ECL Reagent

Cat. No. 345818

Highlights:

• Convenient:
  Just spray…..that’s it! No need for mixing luminol and enhancer solution. No need to pipet or agitate membrane during substrate addition.
• Sensitive:
  As little as 2 pg of protein can be detected.
• Long Lasting:
  Light emission is detected up to 2 hours after the addition of substrate.
• Versatile:
  Can be used with either PVDF or nitrocellulose.

RapidStep™ ECL Reagent is a chemiluminescent system for detection of horseradish peroxidase (HRP) on Western blots. This reagent provides a chemiluminescent signal that is both intense and long-lasting. The unique feature that differentiates this system from other chemiluminescent systems is that the substrate and enhancer solution are premixed in a homogenous, stable solution. A specially designed spray bottle, with a precision misting mechanism, is used to apply the solution directly onto the membrane.

The best feature of RapidStep™ ECL is the convenience and ease of use it provides since no pipeting or mixing is required. After the last wash step in the Western blotting protocol, spray RapidStep™ onto the membrane, just enough to ensure even coverage. Usually this requires between 2 and 5 sprays for a mini-gel precut membrane. Wetting the membrane more than necessary does not provide any advantages; it is advised to spray until the membrane is just moist. Incubate the substrate on the membrane for 1-2 minutes. At this point, remove the excess liquid by touching the membrane against absorbent paper and enclose the membrane in a sheet of plastic wrap, being careful to remove any air bubbles between the membrane and the wrap. Place the wrapped membrane in a film cassette, protein side up and expose to X-ray film for varying amounts of time. Generally expose the membrane for several seconds and 1, 3, 5, and 10 minutes.

In order to obtain low background and crisp signals, optimization must be done with the primary and secondary antibody. Optimizing these variables will reduce non-specific reactions and give strong specific results with an optimal signal-to-background. Researchers have noted that since RapidStep™ is more sensitive than other ECL reagents, it is best to reduce the amount of primary antibody. If you are currently using another ECL system, it will be necessary to adjust experimental conditions with RapidStep™. Once experimental conditions are optimized, however, RapidStep™ delivers superior results.

Superior Sensitivity!

Detection of recombinant Pit-1 using decreasing amounts of protein
Sample: Recombinant Pit-1 protein
Primary Antibody: Anti-Pit-1 Rabbit pAb (1:2000 dilution)
Exposure Time: 5 seconds

Long Lasting Signal!

 Detection of mouse IgG by dot blot Antibody: Anti - mouse IgG HRP (1:5000 diluting) Exposure Time: 3 minutes

RapidStep™ allows for convenience and time-savings, without sacrificing quality. This reagent is versatile for use with nitrocellulose as well as PVDF membranes. In addition, RapidStep™ is highly robust, where light emission can be detected up to 2 hours after addition to the membrane.

Suggestions for Use

• Since RapidStep is sensitive, use less primary and secondary antibody for sharp bands and low background.

• Do not overspray this reagent. Spray only enough to wet the membrane.

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