Bio-Rad Immun-Star AP Chemiluminescence Kits User Manual
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
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
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
References and Acknowledgements ......................26
Note on Electrophoresis and Blotting
Equipment..............................................................27
Appendix 1 Total Protein Detection Procedure ......28
* Note important blocking solution instructions on
page 13. High concentrations of blocker can lead to
high background on blots.
C
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 cm
2
of membrane or approximately 50 mini blots
of ~50 cm
2
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
1
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
3
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
Quantity Shelf
Description Provided Storage Life
Catalog #170-5010/170-5011
Immun-Star Detection Kits
– 2,500 cm
2
• 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
Quantity Shelf
Description Provided Storage Life
Catalog #170-5012
Immun-Star Substrate Pack
– 2,500 cm
2
• Immun-Star chemiluminescent substrate 125 ml 4°C 1 yr
• Immun-Star enhancer** 6.25 ml 4°C 1 yr
Catalog #170-5018
Immun-Star Substrate
– 2,500 cm
2
• Immun-Star substrate 125 ml 4°C 1 yr
Catalog #170-5013/170-5014
Immun-Star Introduction Kits
– 8 mini blots
• Immun-Star chemiluminescent substrate 20 ml 4°C 1 yr
• 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
Blotting Reagents Pack
– 2,500 cm
2
• 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
* 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.
5
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.
6
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 (ddH
2
O) 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.
7
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
8
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