Immun-Star™ AP
Chemiluminescent Protein
Detection Systems
For Use With Nitrocellulose
and PVDF Membranes
Instruction Manual
Catalog #
170-5010, 170-5011, 170-5012,
170-5013, 170-5014, 170-5015,
170-5018, 170-5056, and 170-5057
Table of Contents |
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Section 1 |
Preparation .......................................... |
1 |
1.1 |
Introduction ............................................................ |
1 |
1.2 |
Method Overview .................................................. |
1 |
1.3 |
Immun-Star Products ............................................ |
2 |
1.4 |
Complementary Products ...................................... |
3 |
1.5 |
Storage and Stability of Kit Components ................ |
4 |
1.6 |
Safety Instructions .................................................. |
6 |
Section 2 |
Assay Instructions ................................ |
6 |
2.1 |
Experimental Strategy |
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and General Recommendations ............................ |
6 |
2.2 |
Reagents Flowchart................................................ |
11 |
2.3 |
Working Solutions .................................................. |
12 |
2.4 |
Assay Procedure .................................................... |
15 |
Section 3 |
Troubleshooting .................................. |
19 |
3.1 |
Troubleshooting Guide .......................................... |
19 |
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References and Acknowledgements ...................... |
26 |
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Note on Electrophoresis and Blotting |
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Equipment.............................................................. |
27 |
Appendix 1 Total Protein Detection Procedure ...... |
28 |
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*Note important blocking solution instructions on page 13. High concentrations of blocker can lead to high background on blots.
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Section 1
Preparation
1.1 Introduction
The Immun-Star chemiluminescent detection system is a sensitive, nonisotopic method for immunodetection of specific antigens immobilized on nitrocellulose or PVDF membrane. The system uses the powerful CDP-Star chemiluminescent substrate and enhancer, which is activated by an alkaline phosphatase enzyme conjugate. Drawing on Bio-Rad’s expertise in protein blotting, the substrate and enhancer are incorporated into a sensitive and easy-to-use western blotting detection protocol.
The Immun-Star anti-mouse and Immun-Star anti-rabbit chemiluminescent detection kits provide enough reagents to assay 2,500 cm2 of membrane or approximately 50 mini blots of ~50 cm2 each. The blotting reagents pack provides all the complementary buffer reagents needed for western blotting, matched in quantity to the detection kits. We also offer the Immun-Star Intro chemiluminescent kits, with enough of all reagents needed for 2 large blots or 8 mini blots. The Immun-Star assay kits are for laboratory use only.
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
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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 with nonfat dry milk, or other protein blocking agents.
The membrane with bound antigen is then incubated with a primary antibody specific for the antigen to be detected. The blot is washed to remove unbound antibody and incubated with the respective second antibody, which has been conjugated to alkaline phosphatase (AP). The membrane is then treated with the chemiluminescent substrate alone for PVDF membranes, and with substrate and enhancer for nitrocellulose. The concentrated enhancer is added to the 1x substrate solution and does not add any extra incubation or wash steps. The blot is then used to expose X-ray or instant film, or imaged by an imager capable of detecting chemiluminescent signals, such as the Bio-Rad VersaDoc™ or ChemiDoc™ system.
1.3 Immun-Star Products
Catalog # Description
170-5010 Immun-Star Goat Anti-Mouse -AP Detection Kit 170-5011 Immun-Star Goat Anti-Rabbit -AP Detection Kit 170-5012 Immun-Star AP Substrate Pack
170-5018 Immun-Star AP Substrate
170-5013 Immun-Star Goat Anti-Mouse -AP Intro Kit 170-5014 Immun-Star Goat Anti-Rabbit -AP Intro Kit
2
Catalog # Description
170-5015 Blotting Reagents Pack
170-5056 GAR-AP Blotting Starter Kit
170-5057 GAM-AP Blotting Starter Kit
1.4 Complementary Products
Catalog # Description
Individual Blotting Grade Reagents
170-6518 Goat Anti-Rabbit IgG (H+L)-AP, 1 ml 170-6520 Goat Anti-Mouse IgG (H+L)-AP, 1 ml 170-6521 Goat Anti-Human IgG (H+L)-AP, 1 ml 170-6435 Premixed Tris-Buffered Saline, 10x, 1 L 170-6531 Tween 20, 100 ml
170-6404 Blotting Grade Blocker, nonfat dry milk, 300 g
Blotting Membranes
Nitrocellulose Membrane, 0.45 µm
162-0113 Sheets, 20 x 20 cm, 5
162-0114 Sheets, 9.2 x 15 cm, 10 162-0115 Roll, 33 cm x 3 m, 1 162-0116 Sheets, 15 x 15 cm, 10 162-0117 Sheets, 9 x 12, 10 162-0145 Sheets, 7 x 8.4 cm, 10 162-0148 Sheets, 11.5 x 16 cm, 10
Nitrocellulose Membrane, 0.2 µm
162-0112 Roll, 33 cm x 3 m, 1 162-0146 Sheets, 7 x 8.4 cm, 10 162-0147 Sheets, 13.5 x 16.5 cm, 10
Immun-Blot PVDF Membranes
162-0175 Sheets, 10 x 15 cm, 10
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Catalog # Description
162-0176 Sheets, 20 x 20 cm, 10 162-0177 Roll, 26 cm x 3.3 m, 1 162-0174 Sheets, 7 x 8.4 cm, 10
Blotting Standards
161-0306 Biotinylated Standards, low range, 250 µl
161-0308 Biotinylated Standards Kit (AP), low range
161-0311 Biotinylated Standards, high range, 250 µl
161-0313 Biotinylated Standards Kit (AP), high range
161-0319 Biotinylated Standards, broad range, 250 µl
161-0322 Biotinylated Standards Kit (AP), broad range
161-0363 Precision Plus Protein™ Unstained Standards
161-0382 Precision Protein™ StrepTactin-AP conjugate
170-6533 Avidin-AP, 1 ml
161-0324 Kaleidoscope™ Prestained Standards
161-0325 Kaleidoscope Polypeptide Standards
161-0375 Precision Plus Protein Kaleidoscope Standards 161-0305 Prestained SDS-PAGE Standards, low range 161-0309 Prestained SDS-PAGE Standards, high range 161-0318 Prestained SDS-PAGE Standards, broad range
1.5 Storage and Stability of Components
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Quantity |
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Shelf |
Description |
Provided |
Storage |
Life |
Catalog #170-5010/170-5011 |
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Immun-Star Detection Kits – 2,500 cm2 |
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• Immun-Star chemiluminescent substrate |
125 ml |
4°C |
1 yr |
• Immun-Star enhancer** |
6.25 ml |
4°C |
1 yr |
• AP conjugated antibody* |
0.5 ml |
-20°C |
1 yr |
4 |
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Quantity |
|
Shelf |
Description |
Provided |
Storage Life |
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Catalog #170-5012 |
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Immun-Star Substrate Pack – 2,500 cm2 |
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|
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• Immun-Star chemiluminescent substrate |
125 ml |
4°C |
1 yr |
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• Immun-Star enhancer** |
|
6.25 ml |
4°C |
1 yr |
Catalog #170-5018 |
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Immun-Star Substrate – 2,500 cm2 |
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• Immun-Star substrate |
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125 ml |
4°C |
1 yr |
Catalog #170-5013/170-5014 |
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Immun-Star Introduction Kits – 8 mini blots |
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• Immun-Star chemiluminescent substrate |
20 ml |
4°C |
1 yr |
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• Immun-Star enhancer** |
|
1 ml |
4°C |
1 yr |
• AP conjugated antibody* |
|
0.1 ml |
-20°C |
1 yr |
• TBS (10x) |
|
220 ml |
4°C or RT |
1 yr |
• Nonfat dry milk |
|
2 g |
4°C or RT |
1 yr |
• Tween 20 |
|
2.5 ml |
4°C or RT |
1 yr |
Catalog #170-5015 |
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Blotting Reagents Pack – 2,500 cm2 |
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• 10x TBS |
|
2 x 1 L |
RT |
1 yr |
• Tween 20 |
|
15 ml |
RT |
1 yr |
• Nonfat dry milk blocker |
|
10 g |
RT |
1 yr |
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*Store conjugate at -20°C until first use, then aliquot and store at -20°C. Avoid repeat freeze/thaw cycles. 4°C storage is also acceptable.
**Immun-Star enhancer is used with nitrocellulose membrane only.
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1.6 Safety Instructions
1.Read the entire instruction manual before beginning the assay.
2.Wear gloves and protective clothing, such as a laboratory coat and goggles, when preparing and working with the solutions in the assay. The Immun-Star enhancer contains diethanolamine, which can cause skin and eye irritation. In case of contact, immediately flush the skin or eyes with copious amounts of water for at least 15 min, and remove contaminated clothing.
Note: See Material Data Safety Sheet on diethanolamine for additional information.
3.Work in well-ventilated areas. Avoid inhalation of vapors when handling solutions containing diethanolamine.
4.Do not mouth-pipet any solutions.
Section 2
Assay Instructions
2.1 Experimental Strategy and General
Recommendations
Temperature. All steps are performed at room temperature (22–25°C) unless indicated otherwise in the instructions. If a lower assay temperature is preferred, it is advisable to double the incubation and wash times for each 10°C decrease in temperature.
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Water Purity. Use only deionized distilled water to prepare all solutions. In addition, care should be taken to prevent alkaline phosphatase contamination of assay solutions. Ideally, distilled deionized water (ddH2O) should be autoclaved or sterile-filtered prior to use in buffers and solutions.
Membrane Selection. The Immun-Star assay is specially designed for use with nitrocellulose and PVDF membranes. This kit is not compatible with nylon membrane. The use of Immun-Star enhancer is required when performing blots on nitrocellulose membrane. Longer exposure times will be necessary if the enhancer is not used, with resulting interference from background development. If proteins are transferred to PVDF membrane, use of the enhancer is not necessary. All other steps in the assay procedure remain the same.
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 in buffer is used for antigen detection. A 1:1,000–1:100,000 dilution is used when ascites fluid is the source of antibody. Optimal dilution factors must be determined experimentally. The optimal antibody concentration is usually considered to be the greatest dilution of antibody reagent that still results in a strong positive signal without significant membrane background or nonspecific reactions.
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Blotting Grade Conjugates. The conjugates supplied by Bio-Rad should be used in the concentrations indicated in Section 2.3. Using a conjugate at higher concentrations may result in an overall increase in background without any increase in detection sensitivity.
Washes and Incubations. Continuous gentle agitation should be used during all reactions. For best results, an orbital shaker should be employed to maintain a uniform exposure of the membrane to the solution.
Detergents. Tween 20 is essential in washing to eliminate overall background and nonspecific hydrophobic reactions. At 0.05%–0.1%, Tween 20 will not disrupt binding of primary antibodies to antigens or antigens to the membrane, but will optimize detection sensitivity by eliminating nonspecific reactions. Increased concentrations of Tween 20 (up to 0.3%) can be used if background problems persist. Alternative detergents should not be substituted. The wash between the blocking step and incubation with the first antibody is essential and should not be altered.
Molecular Weight Standards. Biotinylated SDS-PAGE standards and Precision Plus Protein™ unstained standards are recommended for molecular weight determinations with the Immun-Star assays. The Biotinylated SDS-PAGE standards are detected by binding avidin-AP or streptavidin-AP to the biotinylated proteins, which will produce a chemiluminescent signal upon reaction with the substrate. The Precision Plus Protein
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