Western Blot Reagents

Immobilon Western Detection Reagents

Exceptional Performance and Value

Immobilon Western Substrates are optimized for high sensitivity and low background over a broad dynamic range at a considerably lower cost than other chemiluminescent reagents.

Sensitive, Versatile Reagents

The substrates' high signal intensity allow detection of high and low abundance proteins. Antibody dilutions can usually be significantly increased compared to other chemiluminescent substrates, which reduces antibody consumption and assay costs.

Immobilon Western Substrates excel in all Western or dot/slot/spot blotting applications. They are compatible with both PVDF and nitrocellulose membranes, as well as all commonly used buffers and blocking reagents.

Immobilon Western HRP Substrate

High sensitivity with low antibody consumption
Broad dynamic range

Immobilon Western AP Substrate

Higher sensitivity than other AP chemiluminescent substrates
Long signal duration

Detect High and Low Abundance Proteins with One Substrate

	HRP	AP
Emission Duration	>2 hours	AP ³3 days
Primary Detection Method	X-ray film or imaging equipment	X-ray film or imaging equipment
Typical Antibody Dilution*	Primary: 1:1,000–1:20,000 Secondary: 1:20,000–1:200,000	Primary: 1:1,000–1:20,000 Secondary: 1:20,000–1:200,000
Stability	>9 months at time of receipt Working solution: 10 days at 2–8°C	>9 months at time of receipt
Recommended Initial Exposure Time for Film	30 seconds	30 seconds
Membrane Compatibility	PVDF or nitrocellulose	PVDF or nitrocellulose
Storage Conditions	2–8°C	2–8°C

* Due to the higher sensitivity of Immobilon Western Substrates, primary and secondary antibody dilutions can be increased 2 to 5 fold compared to traditional, low sensitivity western substrates.

HRP Substrate Performance

Higher Sensitivity than Traditional HRP Substrates

Detection of transferrin using three HRP substrates on Immobilon-P Membrane (IPVH00010). Two-fold serial dilutions of transferrin were loaded and detected with goat anti-transferrin antibody (dilution 1:10,000) and HRP-conjugated rabbit anti-goat secondary antibody (dilution 1:100,000). All blots were exposed to the X-ray film for 1 minute.

Longer Signal Duration and Higher Intensity

Comparison of signal intensity and duration between western blotting HRP substrates.Each reaction was performed with 0.37 ng of HRP-conjugated rabbit anti-goat antibody (Sigma). Measurements were taken using a Wallac 1420 VICTOR2 multilabel counter (PerkinElmer).

AP Substrate Performance

Higher Sensitivity than Other AP Substrates Detection of transferrin using three AP substrates on Immobilon-P Membrane (Millipore).

Two-fold serial dilutions of transferrin were loaded and detected with goat anti-transferrin antibody (dilution 1:10,000) and AP-conjugated rabbit anti-goat secondary antibody (dilution 1:100,000). Alll blots were exposed to the X-ray film for 1 minute.

Spray & Glow Western Blot Detection Reagent

Spray & Glow is a simple, but novel, enhanced chemiluminescent (ECL) detection reagent with the "at-home" convenience of a spray bottle! Spray the Western membrane and detect your target protein using standard detection methods, without the mixing of substrate and enhancer solutions. Millipore's NEW Spray & Glow™ ECL Western Blotting Detection System will revolutionize Western blotting detection by putting everything you need for a high glow reaction chemiluminescent substrate together in one bottle. Just spray the luminol-based reagent on either PVDF or Nitrocellulose membranes, incubate and detect. It's easy-to-use, long-lasting chemiluminescent signal at a low per-blot cost. The data show higher signal-to-noise ratios when compared to existing mixing of luminol peroxidase formulations or enhanced chemiluminescence reagents on the market! Combined with Millipore's extensive line of primary antibodies, Spray & Glow™ ECL Western Blotting Detection System provides your laboratory with the fast, quality results you require for your research. For more information, please visit www.Millipore.com.

Immunoblot Analysis

Whole adult rat brain lysate was resolved by electrophoresis, transferred to PVDF membrane and probed with anti-GluR2 (Millipore, cat #AB1768). Proteins were detected using an HRP conjugated secondary antibody and Spray & Glow (cat. #17-373, lane 1) or ChemiLucent ECL Detection System (lane 2). Arrow indicates GluR2 (~105 kD).

How does Spray & Glow™TM™ Work?

Hydroxide ion (OH-) generated by conjugated horseradish peroxidase (HRP) in aprotic media result in transitions of luminol to 3'-aminophthalate, which induce emission of 425–510 nm light. To detect the induced emission of 425–510 nm light from the Spray & Glow™ reagent; use either a darkroom and standard X-ray film or a digital imaging technology.

Before ReBot Plus: Western blot probed with rabbit antibodies to peptide from angiotensin II type 1 receptor, 20 second exposure.

After ReBot Plus: Western blot stripped with ReBlot Plus Mild stripping solution.

Reprobe: Western blot reprobed with rabbit antibodies to peptide from glutamate receptor delta 1/2, 20 second exposure.

ReBlot Plus™ Western Blot Recycling Kit

Offers You a Choice of Stripping Solutions

This kit contains two specially formulated antibody stripping solutions (Mild and Strong) that quickly and effectively remove antibodies and their corresponding chemiluminescent signal from membrane blots, without destroying the blotted protein sample.

Stripping and Re-probing of Western Blots Offers Several Advantages

Conserve expensive or limited samples.
Analyze a given blot using several different antibodies.
Re-analyze anomalous results with the same or different antibody.
No-mercaptoethanol in the Stripping Solutions
Save money, reagents and time by reusing the same blot.

Sufficient for 250–500 Membrane Strips or 25–50 Standard Blots (Trial size is sufficient for 50–100 Membrane Strips or 5–10 Standard Blots).

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