Bio-Rad Immun-Blot AP Colorimetric Kits User Manual
Immun-Blot
Assay Kit
Instruction
Manual
®
Table of Contents
Section 1 Preparation .................................................................... 1
1.1 Introduction ................................................................................ 1
1.2 Product Information.................................................................... 2
1.3 Materials Provided with the Immun-Blot Kit; Storage
and Stability of Kit Components ................................................ 4
1.4 Safety Instructions...................................................................... 5
1.5 Solutions..................................................................................... 6
Section 2 Immun-Blot Assay......................................................... 9
2.1 Experimental Strategy and General Recommendations............. 9
2.2 Detailed Assay Procedure........................................................... 11
2.3 Detailed Color Development Procedure..................................... 13
Section 3 Troubleshooting............................................................. 16
3.1 Tests for Monitoring Reagent Activity....................................... 16
3.2 Troubleshooting Guide............................................................... 17
Section 4 References ...................................................................... 21
Section 1
Preparation
1.1 Introduction
The Immun-Blot assay kits are enzyme immunoassay kits
optimized for the detection of specific antigens, at picogram levels,
following electrophoretic blotting or dot blotting to a membrane. The
kits provide all necessary components and chemicals, in an easy-to-use
form, for detection of selected purified proteins using a double
antibody or antibody-binding protein blotting assay.
The Immun-Blot alkaline phosphatase and Immun-Blot horseradish
peroxidase assay systems can be used to detect rabbit, mouse, or human
immune complexes.
antigens or cloned translation products following dot-blotting,
electrophoretic blotting,
or plaque lifts.
The Immun-Blot assay is fast and simple. Antigen is transferred
and bound to the membrane. This transfer can be done electrophoretically, following separation of the antigen in a polyacrylamide or
agarose gel, or passively by either directly spotting the antigen to a
membrane or by overlaying a phage plate or lysed bacterial colony with
nitrocellulose membrane to bind expressed proteins. Following binding
of antigen, the remaining protein binding sites on the membrane
surface are blocked with gelatin or equivalent proteins which will not
react with the primary and secondary antibodies. BSA (not included in
the kit) is used as a blocker when the Bio-Dot
apparatus is used for blotting, since gelatin will obstruct filtration
through blotting membranes.
The membrane with bound antigen is then incubated with first
antibody, specific for the antigen to be detected. The membrane is
1
They will give sensible detection of specific
3-11
filter affinity transfers, 12and in situ colony
®
or Bio-Dot SF
2
1
washed to remove unbound antibody and incubated with the respective
GAR, GAM, or GAH second antibody, or Protein A or Protein G,
which has been conjugated to alkaline phosphatase (AP) or horseradish
peroxidase (HRP). In the case of AP, a color development reagent
containing 5-bromo-4-chloro-3-indoyl phosphate (BCIP) and nitroblue
tetrazolium (NBT) yields purple bands or “dots” on a white
background. The HRP color development reagent, containing 4-chloro1-naphthol and hydrogen peroxidase, also yields purple bands against a
white background.
1.2 Product Information
Catalog
Number Product Description
Immun-Blot Assay Kit
170-6460 Goat Anti-Rabbit IgG (H+L) AP
170-6461 Goat Anti-Mouse IgG (H+L) AP
170-6462 Goat Anti-Human IgG (H+L) AP
170-6463 Goat Anti-Rabbit IgG (H+L) HRP
170-6464 Goat Anti-Mouse IgG (H+L) HRP
170-6465 Goat Anti-Human IgG (H+L) HRP
170-6466 Protein A-HRP
170-6467 Protein G-HRP
Individual Blotting Grade Reagents
170-6518 Goat Anti-Rabbit IgG (H+L) AP Conjugate
170-6520 Goat Anti-Mouse IgG (H+L) AP Conjugate
170-6521 Goat Anti-Human IgG (H+L) AP Conjugate
170-6515 Goat Anti-Rabbit IgG (H+L) HRP Conjugate
170-6516 Goat Anti-Mouse IgG (H+L) HRP Conjugate
172-1050 Goat Anti-Human IgG (H+L) HRP Conjugate
170-6522 Protein A-HRP Conjugate
Catalog
Number Product Description
Individual Blotting Grade Reagents (continued)
170-6425 Protein G-HRP Conjugate
170-6435 Premixed Tris Buffered Saline, 10x, 1 L
170-6431 Horseradish Peroxidase Conjugate Substrate Kit
170-6432 Alkaline Phosphate Conjugate Substrate Kit
170-6537 Gelatin, Blotting Grade, 200 g
170-6531 Tween-20, Blotting Grade, 100 ml
Blotting Membranes
Nitrocellulose Membrane (0.45 µm)
162-0115 Roll, 33 cm x 3 m, 1
162-0145 Sheets, 7 x 8.4 cm, 10
162-0117 Sheets, 9 x 12 cm, 10
162-0114 Sheets, 15 x 9.2 cm, 10
162-0116 Sheets, 15 x 15 cm, 10
162-0113 Sheets, 20 x 20 cm, 5
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
Blotting Media
Supported Nitrocellulose Membrane (0.45 µm)
162-0190 Sheets, 7 x 8.4 cm, 10
162-0191 Sheets, 10 x 15 cm, 10
162-0192 Sheets, 15 x 15 cm, 10
162-0193 Sheets, 20 x 20 cm, 10
162-0194 Roll, 30 cm x 3 m, 1
2
3
Catalog
Number Product Description
Supported Nitrocellulose Membrane (0.2 µm)
162-0195 Sheets, 7 x 8.4 cm, 10
162-0196 Sheets, 15 x 15 cm, 10
162-0197 Roll, 30 cm x 3 m, 1
PVDF Membrane (0.2 µm)
162-0180 Sheets, 10 x 15 cm, 10
162-0181 Sheets, 15 x 15 cm, 10
162-0183 Sheets, 20 x 20 cm, 10
162-0184 Roll, 24 cm x 3.3 m, 1
162-0185 Sheets, 20 x 20 cm, 3
162-0186 Sheets, 7 x 8.4 cm, 10
1.3 Materials Provided with the Immun-Blot Kit
Storage and Stability of Kit Components
Quantity Shelf
Product Description Provided Storage Life
Components supplied in all kits
Tris buffered saline, 10x, pH 7.5 1 L 4 °C >1 yr
Gelatin, Blotting grade 50 g RT >1 yr
Tween-20, Blotting grade 5 ml RT >1 yr
Conjugate solution* 0.5 ml 4 °C 1 yr
Components supplied in alkaline phosphatase kits
AP color reagent A (contains NBT in 10 ml -20 °C 1 yr
aqueous dimethylformamide [DMF],
containing magnesium chloride)
AP color reagent B (contains 10 ml -20 °C 1 yr
BCIP in DMF)
AP color development buffer, 25x 40 ml 4 °C >1 yr
Product Description Provided Storage Life
Quantity Shelf
Components supplied in horseradish peroxidase kits
HRP color regent A (contains 4-chloro- 200 ml -20 °C 1 yr
1-naphthol in diethylene glycol)
HRP color reagents B (contains 10 ml 4 °C 1 yr
hydrogen peroxide)
HRP color development buffer, 10x 100 ml 4 °C >1 yr
Additional components
Citrate buffered saline (CBS), 3x, 100 g RT >1 yr
pH 5.5 (provided in the Protein G HRP
kit only)
* Each kit provides a different conjugate solution. These reagents are shipped
frozen, and can be stored at -20 °C until their initial use. Once thawed, store the
conjugate solution at 4 °C. Avoid repeated freeze-thaw cycles, which will cause
breakdown of the product.
Note: Each kit contains all reagents necessary to complete 200 x 5 ml
assays. An excess of conjugate has been supplied in each kit. BSA must
be substituted for gelatin when blocking the membrane in the Bio-Dot
or Bio-Dot SF microfiltration apparatus.
1.4 Safety Instructions
Read entire instruction manual before beginning the assay.
1. Wear gloves and protective clothing, such as a laboratory coat and
goggles, when preparing and working with the solutions in the
assay. DMF, diethylene glycol, 4-chloro-1-naphthol, and BCIP can
cause skin and eye irritation, and contact should be avoided. In case
of contact, immediately flush the skin or eyes with copious
amounts of water for at least 15 minutes, and remove contaminated
clothing.
4
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