Bio-Rad Immun-Blot Opti-4CN Colorimetric Kits User Manual

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

Table of Contents

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

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 v ersion
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 impro ve­ment of 16–64 fold. The kits provide reagents for ampli­fication and/or detection on 2,500 cm

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, follo wing
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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of membrane.

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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

170-8236 Opti-4CN Detection Kit, Goat-anti-Rabbit

170-8237 Opti-4CN Detection Kit, Goat-anti-Mouse

170-8238 Amplified Opti-4CN Substrate Kit

Opti-4CN substrate, 12.5 ml
Opti-4CN diluent concentrate, 10x, 62.5 ml

Opti-4CN substrate, 12.5 ml
Opti-4CN diluent concentrate, 10x, 62.5 ml
Goat-anti-Rabbit-HRP conjugated secondary
antibody, 0.5 ml

Opti-4CN substrate, 12.5 ml
Opti-4CN diluent concentrate, 10x, 62.5 ml
Goat-anti-Mouse-HRP conjugated secondary
antibody, 0.5 ml

Bio-Rad Amplification Reagent, 53 ml
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

170-8240 Amplified Opti-4CN Detection Kit, Goat-anti-Mouse

Additional required items not provided in these kits

All kits Nitrocelluose or PVDF membrane

170-8235 Blocker

170-8236/7 Blocker

170-8238 Dimethyl sulfoxide (DMSO), 500 ml, sufficient

170-8239/40 Dimethyl sulfoxide (DMSO), 500 ml, sufficient

All components of 170-8238
Goat-anti-Rabbit-HRP conjugated secondary
antibody, 0.5 ml

All components of 170-8238
Goat-anti-Mouse-HRP conjugated secondary
antibody, 0.5 ml

Primary antibody
Tween-20, 10 ml, suf ficient for 2,500 cm
Bovine serum albumin, 7.5 g, sufficient for

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2,500 cm

Buffer
HRP-linked secondary antibody

Buffer

for 2,500 cm
HRP-linked secondary antibody

for 2,500 cm

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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 labora-

tory 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. Av oid 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 an y 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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