Sigma Chemiluminescent Peroxidase Substrate 产品说明书

Chemiluminescent Peroxidase Substrate

Product Codes CPS-1, CPS-1-30, CPS-1-60,

CPS-1-120, and CPS-1-300

Storage Temperature 2-8 °C

TECHNICAL BULLETIN

Product Description

Sigma’s Chemiluminescent Peroxidase Substrate can

be used for the highly sensitive detection of peroxidase

labeled material in a variety of Western blotting

applications. This substrate is an enhanced luminol

product with a stabilized peroxide buffer solution that

provides picogram sensitivity with minimal background

interference.

Components

The Chemiluminescent Peroxidase Substrate is

available in 4 package sizes each containing the

Chemiluminescent Reagent (Product Code C 9107)

and the Chemiluminescent Reaction Buffer

(Product Code C 9232).

Package Size C 9107 C 9232

30 ml 10 ml 20 ml

60 ml 20 ml 40 ml

120 ml 40 ml 80 ml

300 ml 100 ml 200 ml

Precautions and Disclaimer

This product is for laboratory research use only. Please

consult the Material Safety Data Sheet for information

regarding hazards and safe handling practices.

Preparation Instructions

Prepare the Working Solution by mixing 1 part of the

Chemiluminescent Reagent (Product Code C 9107)

with 2 parts of the Chemiluminescent Reaction Buffer

(Product Code C 9232). Mix well and protect from light.

It is recommended to use 0.043 to 0.125 ml per cm

2

of

membrane. For extended signal duration, a 1:1 ratio of

Chemiluminescent Reagent to Chemiluminescent

Reaction Buffer may be used.

Storage/Stability

It is recommended to store the components at 2-8 °C.

The components are stable for a minimum of

18 months when stored in the original container and

protected from light. The Working Solution is stable for

several hours at room temperature when protected from

light.

Procedure

Sigma’s Chemiluminescent Peroxidase Substrate is

very sensitive and great care must be taken to optimize

the individual assay components (antibodies,

conjugates, etc). In a Western blot, an optimized

system is needed to minimize background reactivity

associated with nonspecific immunochemical

interactions. The following is a general guideline for the

use of this product. The protocol starts with a

transferred membrane.

Notes:

• For optimal results, individual assay components

must be optimized for minimal background and

maximal signal.

• This product is designed for use only in Western

blotting.

• All steps below should be performed with slight

agitation on a rocker or an orbital shaker such that

the membrane is freely floating.

• All incubations should be performed at room

temperature.

• Gloves must be worn when working with the

membrane to avoid contamination.

• Azide inhibits horseradish peroxidase (HRP) and

should not be used as a buffer preservative for

assay components.

2

1. Remove membrane from Western blotting

apparatus and wash membrane for 1 minute in

either Tris-buffered Saline with TWEEN



20 (TBST,

Product Code T 9039) or phosphate buffered saline

with TWEEN 20 (PBST, Product Code P 3563).

Note that either a TBS or PBS system can be used

for Western blotting.

2. Block membrane in appropriate blocking agent for

30 minutes. Western Blocker Solution (Product

Code W 0138) is recommended for high sensitivity

detection.

3. Add primary antibody to the blocking agent. The

final concentration of primary antibody in this

solution can range from 0.2-20 µg/ml.

4. Incubate membrane with the primary antibody

solution for at least 30 minutes.

5. Wash with TBST or PBST for 1 minute.

6. Remove TBST or PBST and add at least 10 ml of

appropriate blocking agent to the membrane. Add

secondary antibody; a 1:50,000 to 1:500,000

dilution in blocking agent may be used.

7. Incubate the membrane with the secondary

antibody solution for 30 minutes.

8. Remove blocking solution and wash membrane

5 times for 5 minutes each with TBST or PBST.

9. Remove the membrane from the wash buffer and

drain any excess liquid from the membrane. Keep

the membrane damp; do not let the membrane dry

out.

10. Place the membrane on a flat sheet of plastic wrap

(or on any clean plastic surface).

11. Develop the blots with the Working Solution for

5 minutes.

12. Drain excess substrate and place in holder or

plastic wrap.

13. Expose BioMax light film to the blot. Exposure

times range from 30 seconds to 10 minutes. It is

best to do a quick exposure of 10 to 30 seconds to

determine what exposure time is needed. If the

signal is too intense even at the short exposure

times, let the signal decay from 1 to 8 hours and

then re-expose the film.

Related Products

Product Name Package Size Product Code

TBS

10 packets T 6664

PBS

10 packets P 3813

Western Blocker

Solution

400 ml W 0138

TBS + 3% milk

10 packets T 8793

PBS + 3% milk

10 packets P 2194

PBS + 5% milk

10 packets P 4739

TBS + TWEEN 20

10 packets T 9039

PBS + TWEEN 20

10 packets P 3563

Anti-Mouse HRP

Antibody

2 ml A 9044

TWEEN is a registered trademark of the ICI Group.

RBG/MKS/MAM 1/04

3

Troubleshooting Guide

Problem Type

Too much background

signal observed.

Cause Solution

Not enough wash steps were performed Double the number of washing steps.

at the end of the blotting.

Too much primary antibody used. Lower the amount of primary antibody used

and wash with TBST for 5 minutes instead of

1 minute after the primary antibody

incubation.

Too much secondary antibody used. Lower the amount of secondary antibody

used.

Lower the amount of secondary antibody

used.

Lower the amount of secondary antibody

used.

Lower the amount of primary antibody used

and wash with TBST for 5 minutes instead of

1 minute after the primary antibody

incubation.

Too much secondary antibody used. Lower the amount of secondary antibody

used.

Membrane is stippled. Secondary antibody has some aggregate Filter secondary antibody.

formation.

No signal is seen with Protein levels are too low for detection. Increase exposure time of film and increase

chemiluminescent reaction level of protein loads.

on membrane.

Not enough primary antibody used. Use more primary antibody.

Not enough secondary antibody used. Use more secondary antibody.

Image is reversed on film Too much secondary antibody used.

(dark background and light

bands).

Bands on membrane have Too much secondary antibody used.

brown or yellow tone.

Nonspecific bands show up Too much primary antibody used.

on membrane.

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Sigma-Aldrich, Inc. warrants that its products conform to the information contained in this and other Sigma-Aldrich publications. Purchaser

must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see reverse side of

the invoice or packing slip.