A Calf Intestine Alkaline Phosphatase-biotin conjugate, 600 units per mg protein.Streptavidin-alkaline phosphatase is formed by the conjugation of alkaline phosphatase and streptavidin. It is suitable for the detection of biotinylated antibodies and other biotinylated molecules in immunocytochemical, immunoblotting and ELISA techniques Preparation : Streptavidin-alkaline phosphatase is prepared using a single-step glutaraldehyde coupling reaction to link calf intestinal alkaline phosphatase to streptavidin. Excess coupling reagent is removed using extensive dialysis. The final product concentration is optimized to a standard titre as determined by its binding to biotinylated protein. As a result the final protein concentration may vary minimally between batches Quality control : In the quality control test the conjugate is tested at a range of concentrations in an ELISA test. Plastic microtitration plates are coated with 50ng/well of biotinylated protein in 0.1M carbonate/bicarbonate buffer, pH9.5. Non-specific binding sites are blocked with 0.25%(w/v) gelatin in Tris buffered saline (TBS) pH7.6. The plates are then washed with TBS containing 1%(w/v) TweenTM20. Conjugate is diluted across the plate to 22.5ng/ml in TBS containing 0.25%(w/v) gelatin and the plate incubated at 37 C for 1 hour. The plate is then washed in TBS containing 0.1%(w/v)Tween 20. Substrate solution (p-nitrophenol phosphate in 10mM diethanolamine buffer pH9.5, 5mM magnesium chloride, 100ul) is added to each well and the plate incubated at 37 C for 1 hour. After this time the reaction is stopped with 50ul/well of 10mM ethylenediamine tetraacetic acid (EDTA), and the absorbance at 405nm determined. Formulation : The conjugate is supplied in 2ml of a solution containing 10mg/ml BSA, 50mM Tris-HCl, 2mM MgCl2 and 0.05% sodium azide at pH8.0. Applications : Streptavidin-alkaline phosphatase can be used to detect biotinylated molecules such as antibodies, in a variety of applications, for example, immunocytochemistry, ELISA and immunoblotting Immunocytochemistry : A working dilution of 1:150 in TBS is sufficient for most applications. It is recommended that TBS be used in all dilutions and washings, rather than phosphate buffered saline (PBS), in order to maintain maximal enzyme activity. ELISA : A working dilution of 1:5000 in TBS is sufficient to detect biotinylated protein coated at 16ng/well on a microtitration plate. In the QC laboratories it has been found that a 1:1000 dilution will detect less than 10ng of biotinylated protein using the assay described above. It is important to note that the activity of alkaline phosphatase depends markedly on the substrate buffer used. Studies in our laboratories have shown that, using diethanolamine/HCl pH9.5, the activity is approximately twice that obtained when using glycine/HCl pH9.5. It is therefore recommended that 3mM p-nitrophenol phosphate in 0.1M diethanolamine/HCl pH9.5, 5M magnesium chloride be used. Immunoblotting : A working dilution of 1:3000 (in TBS) is sufficient for most applications. Substrate systems : Immunocytochemistry : New fuchsin method - Prepare the reagent just before use. 1) 20ml, Tris/HCl pH9.0 (0.2M), containing magnesium chloride (5mM) 2) 5mg naphthol-AS-BI-phosphate 3) 100ul dimethylformamide 4) 250ul sodium nitrite (0.6M) - must be freshly made 5) 250ul new fuchsin (4%(w/v) in 2M HCl). This can be stored at 2-8 C. Dissolve 2 in 3, mix 4 and 5, then add both solutions to 1, to form a yellow coloured solution, and mix well. Apply to the section and incubate for 5-30 minutes at room temperature. Slides may be counterstained, dehydrated, cleared and mounted in a permanent mounting media. Note: If cryostat sections are being immunostained, it may be necessary to inhibit endogenous alkaline phosphatase by adding 1mM levamisole to the final incubating medium. This will inhibit all alkaline phosphatase except the intestinal enzyme. Addition of levamisole does not alter the pH of the incubating medium. ELISA : Nirophenol phosphate methods- prepare the reagent just before use. Dissolve 0.1mM p-nitrophenol phosphate in 0.1M diethanolamine/HCl pH9.5, containing 5mM magnesium chloride. Incubate for 1 hour at room temperature. The reaction may be stopped with 0.1M ethylene diamine tetra acetic acid (EDTA). The yellow reaction product absorbs maximally at 405nm. Immunoblotting : NBT/BCIP method- prepare the reagent just before use. 1) 10ml 0.1M diethanolamine/HCl pH9.5 containing 5mM magnesium chloride. 2) 3.3mg Nitro blue tetrazolium (NBT) dissolved in 44ul of 70% (w/v) dimethylformamide. 3) 1.65mg bromo-chloro-indolyl phosphate (BCIP) in 33ul of 100%(w/v)dimethylformamide. Add both 2 and 3 to solution 1, mix well and use immediately. Incubate at room temperature until a reaction product is seen.
