4100130c.qxd 4/9/99 8:29 AM Page Cvr2 Opti-4CN™ Substrate Kit 170-8235 Opti-4CN Detection Kit, Goat-anti-Rabbit 170-8236 Opti-4CN Detection Kit, Goat-anti-Mouse 170-8237 Amplified Opti-4CN Substrate Kit 170-8238 Amplified Opti-4CN Detection Kit, Goat-anti-Rabbit 170-8239 Amplified Opti-4CN Detection Kit, Goat-anti-Mouse 170-8240 Instruction Manual For Technical Service Call Your Local Bio-Rad Office or in the U.S.
4100130c.qxd 4/9/99 8:29 AM Page i Table of Contents Section 1 Preparation............................................1 1.1 1.2 1.3 1.4 1.5 Introduction.....................................................1 Method Overview .......................................... 1 Kit Components ............................................. 2 Product Storage and Stability..........................3 Safety Instructions ......................................... 4 Section 2 Protocol.................................
4100130c.qxd 4/9/99 8:29 AM Page 1 Section 1 Preparation 1.1 Introduction Opti-4CN is an improved and more sensitive version of the colorimetric horseradish peroxidase (HRP) substrate, 4-chloro-1-naphthol (4CN). Opti-4CN may be used simply as a replacement for 4CN resulting in a 4–8 fold increase in detection sensitivity.
100130c.qxd 4/9/99 8:29 AM Page 2 The membrane with bound antigen is then incubated with a primary antibody specific to the antigen of interest. The blot is washed to remove unbound antibody, incubated with a secondary antibody linked to HRP, and then washed again to remove unbound secondary antibody. If there is no amplification involved (i.e.
4100130c.qxd 4/9/99 8:29 AM Page 4 Antibodies are provided in 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.2, with 1% (w/v) bovine serum albumin and 0.01% thimerosal (sodium ethylmercurithiosalicylate) as a preservative. Avoid freeze-thaw cycles of antibody conjugates which will cause breakdown of product. For best results, aliquot conjugates in appropriate amounts and store at -20 °C. 1.5 Safety Instructions Please read the entire instruction manual before beginning the protocol. 1.
4100130c.qxd 4/9/99 8:29 AM Page 6 ascites fluid is the source of antibody. Optimal dilution factors must be determined experimentally. The optimal antibody concentration is usually considered the greatest dilution of antibody reagent still resulting in a strong positive signal without membrane background or non-specific reactions. Secondary Antibody Conjugates. The protocols in this manual were worked out using Bio-Rad Blotting Grade secondary antibody conjugates diluted as described below.
4100130c.qxd 4/9/99 8:29 AM Page 8 3% Blocker. While vigorously stirring 665 ml of PBST, very slowly add 20 g powdered blocker. Add the powder a little bit at a time over a period of 30–45 minutes. Continue stirring another 30–60 minutes after all the powder has been added. Slowly warming the solution to 55 °C will help put the blocker into solution, but do not overheat. This can be accomplished by placing the flask with the blocker solution inside a beaker on top of a magnetic stirrer/heater.
4100130c.qxd 4/9/99 8:29 AM Page 10 Colorimetric Detection 14.Mix one part Opti-4CN diluent concentrate with nine parts ddH2O. Prepare 0.25 ml per cm2 of membrane. 15.Add 0.2 ml of Opti-4CN substrate per 10 mls of diluent (e.g., combine 1 ml Opti-4CN diluent concentrate with 9 ml ddH2O and 0.2 ml Opti-4CN substrate). Mix well and pour onto membrane. 16.Incubate membrane with gentle agitation in the substrate for up to 30 minutes or until the desired level of sensitivity is attained. 17.
4100130c.qxd 4/9/99 8:29 AM Page 12 3. Blocking step. Immerse the membrane at a 45° angle into the blocking solution. Gently agitate the solution using a rocking platform and incubate for an hour or more. Best results for amplification are attained with a 3% solution of the blocker provided with the kits (170-8238/39/40). For non-amplified applications, 5% blotto is an acceptable alternative. 4. Wash. Decant the blocking solution and add PBST to the membrane. Wash for 5 minutes.
4100130c.qxd 4/9/99 8:29 AM Page 14 12. Wash. Pour off the streptavidin-HRP solution and add PBST. Wash the membrane for 5 minutes with gentle agitation. Repeat the wash step with fresh PBST. Colorimetric Detection 13. Prepare 0.25 ml of Opti-4CN diluent solution per cm2 of membrane by first mixing 1 part Opti-4CN diluent concentrate with 9 parts ddH2O. For each 10 ml of diluent, add 0.2 ml of Opti-4CN substrate and mix well. The solution will become cloudy upon mixing. 14.
4100130c.qxd 4/9/99 8:29 AM Page 16 10. Wash 2x in PBST for 5 minutes each time. 11. Incubate membrane in diluted BAR for 10 minutes. 12. Wash 2–4x in 20% DMSO/PBST for 5 minutes each time. 13. Wash 1–2x in PBST for 5 minutes each time. 14. Incubate membrane in diluted streptavidin-HRP for 30 minutes. 15. Wash 2x in PBST for 5 minutes each time. Section 4 Troubleshooting Guide Problem Probable Cause Recommended Solution 1. No reaction or weak signal. a. Exposure time was too short. i.
4100130c.qxd 4/9/99 8:29 AM Page 18 Troubleshooting Guide (continued) Problem Probable Cause Recommended Solution c. Conjugate is inactive. i. Store the conjugate at the proper temperature. Avoid repeated freeze-thaw cycles. Troubleshooting Guide (continued) Problem Probable Cause Recommended Solution d. Little or no antigen is bound to the membrane. i. Tween-20 may wash bound antigen from the membrane. Eliminate Tween-20 from the assay (except the wash after the blocking step). ii.
4100130c.qxd 4/9/99 8:29 AM Page 20 Troubleshooting Guide (continued) Probable Cause Recommended Solution e. Primary antibody is not specific or does not recognize denatured antigens (common with monoclonals). i. Loss of reac tivity may have occurred during electrophoretic transfer. Pretest the reactivity of the antibody against the antigen by a dot blot. 2. High background. a. Exposure time was too long. i. Decrease exposure time. b. PBST washes after transfer were omitted or insufficient. i.
4100130c.qxd 4/9/99 8:29 AM Page 22 Troubleshooting Guide (continued) Problem Probable Cause Recommended Solution h. BAR concentration was too high. i. Dilute BAR further. i. Streptavidin-HRP concentration was too high i. Dilute StreptavidinHRP further.
4100130c.qxd 4/9/99 Catalog Number 8:29 AM Page 24 Product Description Supported Nitrocellulose Membranes (0.2 micron) 162-0095 Sheets, 7 x 8.4 cm, 10 162-0096 Sheets, 15 x 15 cm, 10 162-0097 Roll, 33 cm x 3 m, 1 PVDF Membranes (0.2 micron) 162-0186 Sheets, 7 x 8.4 cm, 10 162-0180 Sheets, 10 x 15 cm, 10 162-0181 Sheets, 15 x 15, 10 162-0182 Sheets, 20 x 20 cm, 10 162-0185 Sheets, 20 x 20 cm, 3 162-0184 Roll, 24 cm x 3.
