De staining Solution Methanol 30 Glacial acetic acid 10 Distilled water 60

De staining solution methanol 30 glacial acetic acid

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De-staining Solution Methanol 30 % Glacial acetic acid 10 % Distilled water 60 % Preservative Solution Glacial acetic acid 7 % Distilled water 93 % 13. ELISA REAGENTS AND BUFFERS A. Coating Buffer Tris buffer 0.1 M, p H 7.0 to 9.0 Or 0.05 M carbonate buffer, p H 9.6 with 0.02 % NaN 3 (Sodium azide) or 0.01 M PBS p H 7.4 with 0.02 % NaN 3 (Sodium azide) For better coating of antibodies and protein antigens : Dilute serum with equal volume of 0.1 M glycine-HCl buffer, p H 2.5 with 0.1 M NaCl, incubate for 10 min, and neutralize with 0.1 M Tris p H 9.0 Or Make antigen in 0.05 M glycine-HCl buffer, p H 2.5 with 0.1 M NaCl, incubate for 10 min, and neutralize with 0.05 M Tris p H 9.0 For lipid/ surface antigens, make suspension of antigen in sodium deoxycholate (1 mg/ml) solution before coating. b. Washing buffer Phosphate buffered saline ( p H 7.2) 1000 ml Tween-20 (Polysorbate) 0.5 ml Sodium azide 0.2 gm Stored at 4°C.
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16 C. Blocking solution Gelatin 1.0 g PBS 100 ml Or 2 % Bovine serum albumen in water with 0.02 % NaN 3 (Sodium azide) or 5% Skim Milk in water with 0.02 % NaN 3 (Sodium azide) D. Substrates for Horse radish peroxidase (HRPO) conjugate: 1. Ortho-phenylene-diamine (0.5 mg/ml) in Citrate-phosphate buffer, 0.1 M, p H 4.6 - 5.0), made fresh and add H 2 O 2 (of 30 %, 5 μl per 30 ml) just before use, avoid exposure to direct light. Read at 492 nm. Buffer Stock Solution A Citric acid (C 6 H 8 O 7 .H2O) 2.10 g in Distilled water 100 ml Buffer Stock Solution B Sodium citrate (Na 3 C 6 H 5 O 7 .2H 2 O) 2.94 g in Distilled water 100 ml Working Solution : 25.5 ml of solution A and 24.5 ml of solution B were mixed just before use. Enzyme activity arrester (1 M, H 2 SO 4 ) Concentrated sulphuric acid 27.77 ml Distilled water 72.33 ml B. Tetramethyl benzidine (TMB): Dissolve 10 mg TMB in 1 ml DMSO and then dilute with 0.1 M sodium acetate citric acid buffer p H 6.0, add 30 μl H 2 O 2 (30 %), read at 450 nm, bright yellow colour after adding sulfuric acid. Enzyme activity arrester (2 M, H 2 SO 4 ) 3. 5-aminosalycilic acid Add 100 mg in 100 ml of 0.01 M Sod. Phosphate buffer, pH 6.0 having 1mM EDTA, add 20 μl H 2 O 2 (30 %), read at 450 nm, brown colour after adding NAOH. Enzyme activity arrester (1 M, NaOH) e. Substrate for Alkaline phosphatase conjugate 1. p-Nitrophenylene phosphate : Substrate solution (1 mg/ml) is made in 0.05 M carbonate buffer p H 9.8 with 0.001 M MgCl 2 . Read at 400 nm. Enzyme activity arrester (1 M, NaOH) Or p-Nitrophenylene phosphate solution (1 mg/ml) can be made in 0.05 M diethanolamine buffer p H 9.8. Read yellow colour at 405 nm.
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17 Diethanolamine buffer: Diethanolamine, 9.7 ml; Water, 80 ml; Sodium azide 0.02 gm. Adjust p H with 1M HCl to 9.8. Final volume is made with distilled water to 100 ml. Enzyme activity arrester (3 M, NaOH) 14. SOLUTIONS FOR ESTIMATION OF PROTEIN A . Modified Lowry’s Method (for 0.1 to 1 mg / ml protein) Solution A:1% CuSo 4 .5H 2 O 1 g/ 100 ml distilled water Solution B: 2% sodium potassium tartarate 1 g/ 50 ml distilled water Solution C: 0.2 M, NaOH 0.8 g/100 ml Solution D: 4% Na 2 CO 3 4 g/100 ml Reagent Mixtures Solution E: 49 ml each of solution C and solution D and 1 ml each of solution A and solution B were mixed prior to use.
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