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for immunochemistry
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Pack Qty.
DAB substrate kit, HRP substrate kit
Storage temp. +4 °C
Transport temp. cooled

Substrate kit for detection of HRP activity on blot membranes and slides.
One kit is sufficient for staining of approx. 44 minigel blot membranes or 500 slides.

DAB substrate kit for detection of peroxidase (HRP) activity on immunoblots, immunohisto- and cytochemical slides and during in situ hybridization.
Product details

€213.95/Pack Qty. 

excl. VAT. | 1 kit per Pack Qty.

Art. No. 9202.1

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Product details

DAB substrate kit for detection of peroxidase (HRP) activity on immunoblots, immunohisto- and cytochemical slides and during in situ hybridization.

ROTI®DAB Kit contains substrate, buffer solution and activation reagent, therefore including all three solutions necessary for HRP detection reaction. Application is very simple - all three kit solutions are diluted equally in water, and the blotting filter membrane, or the slides, are then incubated in this substrate solution. Since the solutions are delivered in drop dispensers, dosing is most convenient.

ROTI®DAB Kit for immunochemistry

DAB is oxidized by HRP, forming a brown precipitate, which is insoluble in aqueous and organic solvents. This precipitate can be detected in visible light and does not bleach during long-term storage.

Figure: Immunohistochemical staining of mouse small intestines with an anti-PCNA antibody. Minuscules: pure DAB substrate, capitals: detection supplemented by DAB enhancer (A: NiCl2, B: DAB Metal Enhancer, Art. No. 9204).
A/a: Basic method using 0.6 mg/ml DAB in Tris-buffer.
B/b: Detection performed with the ROTI®DAB Kit.

Directions for use

Addition of the DAB Metal Enhancer (Art. No. 9204) doubles the signal strength. Slides may be permanently mounted in hydrophilic, or, after dehydration, in hydrophobic mounting medium.

The kit contains

DAB solution (Art. No. 9869), DAB buffer (Art. No. 9870), and Activation reagent (Art. No. 9871) in dropper bottles. Contents of this Kit may not be bought separately.

Technical Information
Application Western, in situ, Histochemistry 
Enzyme Horseradish Peroxidase 
Detection Colour (brown) 
Signal product Precipitate (Blot) 
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Order No. Pack Qty. Pack. Packaging Price Quantity
9202.1 1 kit cardboard for ca. 40 assays, in dropper bottles


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Downloads / MSDS

General information

Assay protocol for use of BCIP/NBT in immunoblot procedures:

Stock solutions (All solutions are stable for at least 1 year at 4 °C):
0.5 g NBT in 10 ml 70 % dimethylformamide;
0.5 g BCIP (p-toluidine salt) in 10 ml 100 % dimethylformamide.
Incubation buffer for alkaline phosphatase:
100 mM NaCl, 5 mM MgCl2,100 mM Tris (pH 9.5).
Fresh substrate solution: 66 μl NBT stock solution + 10 ml incubation buffer, mix well, add 33 μl BCIP stock solution. Use within 1 hour.
Blot development: Approx. 10 ml substrate solution per 15 x 15 cm2 membrane surface. Develop at room temperature until bands become visible (approx. 30 mins).
Reaction stop: Rinse with PBS/20 mM EDTA.

Detection of peroxidase activity (Immunoassay):

Dissolve 1 mg TMB in 0.1 ml dimethylsulfoxide (Art. No. 4720); add 9.9 ml of a 0.1 M sodium acetate solution (pH 6.0) (Powder: Art. No. 6779), filter and add H2O2 (Art. No. 8070) (final concentration 0.01 %).

Always prepare freshly!

Incubation 10-30 mins at room temperature (approx. 50 μl per microtitre well; finally add 50 μl 1 M H2SO4, Art. No. X873, per well).
Photometric quantitation at 450 nm.

Reference: Bos E.S. et al. (1981) J. Immunoassay 2, (3/4), 187.

Certificates of Analysis

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The following analysis certificates have been found:

Type analysis

Application test (Blotting)complies
RNAse≤1 ppb
DNAse≤1 ppm