6- Chloro-l-naphthol

There are other chromogens that can be employed when using peroxidase enzymes. The compound 4-chloro-l-naphthol is one that is often used in... 

Enzyme-Immunoassay. Fish tissue samples for testing were cut into uniform 3mm thick slices with parallel razor blades mounted on a handle. Four discs were then punched out from each slice with a stainless steel borer, 3-mm in diameter, and each disc was placed in a well of a 96-well polystyrene microtiter plate (Flow Laboratories, Inc., Hamden, CT). Samples were washed once with 0.2 ml Tris buffer. After the wash solution was aspirated, each sample was fixed in 0.2 ml of 0.3% H O -methanol fixative for 30 min. at room temperature. Samples were then transferred to clean wells and 0.2 ml of a 1 100 dilution of sheep-anti-ciguatoxin-horseradish peroxidase conjugate in Tris buffer was added to each well. The plate was then incubated at room temperature for 1 hr. The sheep-anti-cigua-toxin-horseradish peroxidase was removed by aspiration, and the tissues were immersed for 5 min. in 0.2 ml Tris buffer. Each sample was transferred to clean wells and incubated for 5 min. at room temperature with 0.2 ml of 4-chloro-l-naphthol substrate. The final steps involved removal of the tissue and addition of 0.015 ml of 3 M sodium hydroxide to stop the enzymatic reaction. Absorbance readings at 405 nm of each well were obtained in the Titertek Multi-skan (Flow Laboratories, Inc., Hamden, CT). 

Chloro-l-naphthol (4-CN) and diaminobenzidine (DAB) are used in Soln. B staining with tetramethylbenzidine (TMB) is done in Soln. G at RT for 10-20 min. 

Chloro-l-naphthol [604-44-4] M 178.6, m 116-117 , 120-121 . Crystd from EtOH or chloroform. 

Con A-HRP conjugate, diluted in 10% BSA solution Substrate solution 4-chloro-l-naphthol in methanol H202, 30% in water... 

Chloro-l-naphthol This substrate for the peroxidase is prepared as stock solution of 0.3% in methanol Add 0 6 mL of the stock solution and 2 pL of H202 (30%) to 9.4 mL of distilled water just before use... 

CN visualization solution Mix 20 ml ice-cold methanol with 60 mg 4-chloro-l-naphthol (4CN). Separately mix 60 pi of 30% (w/v) H202 with 100 ml TBS (see recipe) at room temperature. Rapidly mix the two solutions and use immediately. 

CMOS complementary metal oxide semiconductor 4-CN 4-chloro-l-naphthol Co cobalt... 

Mixture for chemiluminescence detection (10 mL) 5 mg 4-chloro-l-naphthol dissolved in 1.7 mL methanol, 2.5 mL 200 mM Tris-HCl pH 7.4 (24.2 g/L), 100 mg NaCl 5.8mLH20,5 pL 30% H202. First, dissolve the NaClin the above amount of water and Tris-buffer (pH 7.4), followed by adding the methanolic chloronaphthol solution. Shortly before use mix this solution with the hydrogen peroxide. 

After blocking the nitrocellulose transfer with 3% (w/v) bovine serum albumin (BSA) in Tris-buffeied saline (TBS), first antibody should be added at a concentration no greater than a 1 1000 dilution. Incubation with first antibody should proceed for a minimum of 1 hr at room temperature, or it can be allowed to continue overnight at 4°. After washing 3 times with TBS, horseradish peroxidase-conjugated second antibody is added (1 1000 in 3% (w/v) BSA-TBS) for 1 hr at room temperature. After 3 more washes with TBS (the final wash with 10% methanol added), 4-chloro-l-naphthol is used to visualize the bound antibodies. 

Horseradish peroxidase 4-Chloro-l-naphthol (CN) Blue/purple... 

Chloro-l-naphthol—This reagent can be purchased from Pierce Chemical Co. (catalog 34010). See the protocol for the preparation of this substrate solution. 

HRP- ubstrate solution Dissolve 120 mg of 4-chloro-l-naphthol in 40 mL of methanol, add 200 mL of TBS and 80 i,L of 30% (v/v) hydrogen peroxide. Make immediately prior to development of the Western blot I-protein A Obtained commercially in buffered aqueous solution, specific activity >30 mCi/mg, stored at 4°C with lead shielding. Use 5... 

Th e most commonly employed de tecdon system is probably that of horseradish peroxidase-conjugated second andbody, in which the enzyme converts a colorless substrate (such as Odianisidine, 3,3-diamino benzidine or 4chloro-l-naphthol) to a colored product, which then becomes irreversibly bound to the membrane. The best substrate, both in terms of chemical safety and sensidvity of detecdon, is 4-chloro-l-naphthol, which wll roudnely detect less than 100 pg of protein (21). 

Chloro-l-naphthol. The reagent should be discarded when crystals, originally white, turn to grey. It is stable for a few months at-20°C when stored desiccated. It can also be stored as a solution in ethanol at-20 C. 

Color reagent solution 60 mg of 4-Chloro-l-naphthol is dissolved in 20 mL of ice-cold 95% ethanol and added to 100 mL of TBS containing 60 i,L of 30% H2O2. The reagent should be prepared Just before using. 

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