Opti-4CN™ Substrate Kit
170-8235
Opti-4CN Detection Kit, Goat-anti-Rabbit
170-8236
Opti-4CN Detection Kit, Goat-anti-Mouse
170-8237
Amplified Opti-4CN Substrate Kit
170-8238
Amplified Opti-4CN Detection Kit, Goat-anti-Rabbit
170-8239
Amplified Opti-4CN Detection Kit, Goat-anti-Mouse
170-8240
Instruction Manual
For Technical Service
Call Your Local Bio-Rad Office or
in the U.S. Call 1-800-4BIORAD
(1-800-424-6723)
Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547
4100130 Rev C
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Table of Contents |
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Section 1 |
Preparation............................................ |
1 |
1.1 |
Introduction..................................................... |
1 |
1.2 |
Method Overview .......................................... |
1 |
1.3 |
Kit Components ............................................. |
2 |
1.4 |
Product Storage and Stability.......................... |
3 |
1.5 |
Safety Instructions ......................................... |
4 |
Section 2 |
Protocol................................................. |
5 |
2.1 |
Experimental Strategy and General |
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Considerations................................................. |
5 |
2.2 |
Reagent Preparation ........................................ |
7 |
2.3 |
Quick Guide .................................................... |
9 |
2.4 |
Detailed Protocol .......................................... |
10 |
Section 3 |
Alternative Protocol............................ |
14 |
Section 4 |
Troubleshooting Guide ....................... |
17 |
Section 5 |
Ordering Information ........................ |
22 |
This product is covered by U.S. Patent 5,583,001 and pending patent applications. Purchase of this product includes a license for use in non-commercial research applications only.
Section 1
Preparation
1.1 Introduction
Opti-4CN is an improved and more sensitive version of the colorimetric horseradish peroxidase (HRP) substrate, 4-chloro-1-naphthol (4CN). Opti-4CN may be used simply as a replacement for 4CN resulting in a 4–8 fold increase in detection sensitivity. When used in conjunction with the signal amplification reagents in the Amplified Opti-4CN substrate and detection kits, sensitivity may be improved another 4–8 fold, resulting in an overall improvement of 16–64 fold. The kits provide reagents for amplification and/or detection on 2,500 cm2 of membrane.
1.2 Method Overview
The first step in western blotting is the transfer of antigen onto a solid support membrane by one of several methods. The transfer can be done electrophoretically, following separation of the antigen in a polyacrylamide or agarose gel, passively by directly spotting the antigen onto a membrane, or by vacuum filtration using a microfiltration apparatus. Following antigen binding, the remaining protein binding sites on the membrane surface are blocked to minimize non-specific interactions.
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The membrane with bound antigen is then incubated with a primary antibody specific to the antigen of interest. The blot is washed to remove unbound antibody, incubated with a secondary antibody linked to HRP, and then washed again to remove unbound secondary antibody. If there is no amplification involved (i.e., catalog numbers 170-8235 through 170-8237), then the blot is incubated in the Opti-4CN substrate for up to 30 minutes, or until the desired sensitivity is attained. If the signal is to be amplified, the blot is incubated in the Bio-Rad amplification reagent (BAR), washed, incubated in streptavidin-HRP, and washed again before being incubated in the Opti-4CN substrate.
1.3 Kit Components
170-8235 |
Opti-4CN Substrate Kit |
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Opti-4CN substrate, 12.5 ml |
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Opti-4CN diluent concentrate, 10x, 62.5 ml |
170-8236 |
Opti-4CN Detection Kit, Goat-anti-Rabbit |
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Opti-4CN substrate, 12.5 ml |
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Opti-4CN diluent concentrate, 10x, 62.5 ml |
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Goat-anti-Rabbit-HRP conjugated secondary |
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antibody, 0.5 ml |
170-8237 |
Opti-4CN Detection Kit, Goat-anti-Mouse |
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Opti-4CN substrate, 12.5 ml |
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Opti-4CN diluent concentrate, 10x, 62.5 ml |
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Goat-anti-Mouse-HRP conjugated secondary |
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antibody, 0.5 ml |
170-8238 |
Amplified Opti-4CN Substrate Kit |
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Bio-Rad Amplification Reagent, 53 ml |
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Streptavidin-HRP, 0.5 ml |
Blocker, 20 g
PBS, powder to make 1 liter of 10x PBS 2x Amplification diluent, 106 ml Opti-4CN substrate, 12.5 ml
Opti-4CN diluent concentrate, 10x, 62.5 ml
170-8239 |
Amplified Opti-4CN Detection Kit, Goat-anti-Rabbit |
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All components of 170-8238 |
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Goat-anti-Rabbit-HRP conjugated secondary |
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antibody, 0.5 ml |
170-8240 |
Amplified Opti-4CN Detection Kit, Goat-anti-Mouse |
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All components of 170-8238 |
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Goat-anti-Mouse-HRP conjugated secondary |
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antibody, 0.5 ml |
Additional required items not provided in these kits
All kits |
Nitrocelluose or PVDF membrane |
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Primary antibody |
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Tween-20, 10 ml, sufficient for 2,500 cm2 |
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Bovine serum albumin, 7.5 g, sufficient for |
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2,500 cm2 |
170-8235 |
Blocker |
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Buffer |
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HRP-linked secondary antibody |
170-8236/7 |
Blocker |
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Buffer |
170-8238 |
Dimethyl sulfoxide (DMSO), 500 ml, sufficient |
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for 2,500 cm2 |
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HRP-linked secondary antibody |
170-8239/40 |
Dimethyl sulfoxide (DMSO), 500 ml, sufficient |
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for 2,500 cm2 |
1.4 Product Storage and Stability
The kit is shipped at 4 °C. Store the unopened kit at 4°C. Powdered blocker, powdered PBS and PBS solutions may be stored at room temperature; all other components are stored at 4 °C. After being put into solution, the blocker should be stored at 4 °C. All kit components are guaranteed for 1 year from the time of receipt.
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Antibodies are provided in 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.2, with 1% (w/v) bovine serum albumin and 0.01% thimerosal (sodium ethylmercurithiosalicylate) as a preservative. Avoid freeze-thaw cycles of antibody conjugates which will cause breakdown of product. For best results, aliquot conjugates in appropriate amounts and store at -20 °C.
1.5 Safety Instructions
Please read the entire instruction manual before beginning the protocol.
1.Wear gloves and protective clothing, such as laboratory coats and goggles when preparing and working with the solutions in the protocol. DMSO is an irritant; it is a colorless liquid which is easily absorbed through the skin and mucous membranes. Avoid skin contact with DMSO and inhalation of DMSO mist. Wash exposed skin thoroughly with soap and water.
Note: See Material Data Safety Sheet (MSDS) on DMSO for additional information.
2.Work in well-ventilated areas. Avoid inhalation of vapors when working with solutions containing DMSO.
3.Do not mouth pipet any solution.
Section 2
Protocol
2.1 Experimental Strategy and General
Considerations
Temperature. All steps are performed at room temperature (22–25 °C).
Making Solutions. Use only deionized, distilled water to prepare solutions. 1x PBST solutions should be sterile filtered. Do not use azide as a preservative in any solution.
Membrane Selection. This kit has been optimized for detection on pure nitrocellulose membranes and performs equally well with supported nitrocellulose membranes. However, due to the way in which they are manufactured, the surface of some supported nitrocellulose membranes take on a ‘wavy’ appearance that can result in less pleasing images than the pure nitrocellulose membranes which remain flat throughout the process. The kit may also be used to detect proteins bound to PVDF.
Primary Antibody. Generally when serum or tissue culture supernatants are the source of primary antibody, a 1:100–1:1,000 dilution of the primary antibody in buffer is used for detection of antigens on the membrane surface. For chromatographically purified monospecific antibodies, a 1:500–1:10,000 dilution is used for antigen detection. A 1:1,000–1:100,000 dilution is used when
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