TECHNICAL INFORMATION

Transcription

1 MP Biomedicals, LLC Fountain Parkway Solon, Ohio TECHNICAL INFORMATION Telephone: 440/ Toll Free: 800/ Fax: 440/ mailto: web: Catalog Number: , , , , , , , Aurora Western Blot Chemiluminescent Detection System 1. Introduction The Aurora Western Blot Chemiluminescence Detection System is designed for the rapid, sensitive nonisotopic identification of proteins and haptens immobilized on membranes. Proteins may be transferred to the membrane following gel electrophoresis, as in standard Western blotting protocols, or bound directly to the membrane in a dot or slot blot format. The Aurora system utilizes enzyme-linked immunodetection of antigen specific antibodies with secondary antibodies conjugated to alkaline phosphatase. Highly specific anti-rabbit, anti-mouse, anti-rat and anti-human IgG-alkaline phosphatase conjugates have been selected by MP for sensitivity and low non-specific binding. The use of enzyme conjugates has many advantages over the use of radioactive labels, including stability, safety and convenience. Alkaline phosphatase is the preferred label in our system due to its stability, insensitivity to azide reagents, and high sensitivity of detection when coupled with the chemiluminescent substrate Starlight. Starlight substrate is a direct chemiluminescent substrate for alkaline phosphatase. Upon dephosphorylation, Starlight decomposes producing a prolonged emission of light which may be imaged on X-ray or instant photographic film. In addition, Starlight provides a permanent record of results that does not fade over time as often occurs with colorimetric systems. 2. Kit Components Aurora Western Blotting kits are available in 2 sizes and sufficient for either 10 or (10 cm x 10 cm). The reagents included in each kit are described below. 1. Chemiluminescent Substrate: Ultra-Pure Starlight chemiluminescent substrate is supplied as a 0.25 mm ready-to-use solution. Store at 2-8 C in the dark. 2. Aurora Blocking Reagent: This protein-based membrane blocking reagent is supplied as a dry powder. Aurora Blocking Reagent is a highly purified casein which has been screened for low alkaline phosphatase contamination and has been shown to be an efficient blocking reagent. Store dry at room temperature. 3. Opti-Membrane Reagent: This chemiluminescent enhancing material is supplied in a 5 ml vial of liquid concentrate (10 mg/ml). Opti-Membrane provides signal enhancement with Nitrocellulose and PVDF membranes. Store at 2-8 C X Assay Buffer: 200 mm Tris-HCl, ph 9.8, 10 mm MgCl2. Dilute 1:10 with deionized water. Store at +4 C. 5. Antibody Conjugate: Each Aurora Western Blot Kit contains one vial of one of the following secondary antibodies conjugated to alkaline phosphatase. Goat anti-rabbit IgG (H&L) having minimal cross-reaction to human serum proteins. Goat anti-mouse IgG and IgM (H&L) having minimal cross-reaction to human, bovine and horse serum proteins. Goat anti-human IgG (H&L). Goat anti-rat IgG and IgM (H&L) These antibodies are supplied as concentrates in 50% glycerol. Store at 2-8 C or optimally at -20 C. 6. Development Folders: 14 cm x 19 cm clear polypropylene sheets for membrane blot handling during imaging. 3. Selection and Preparation of Membranes Enzyme Kinetics

2 The procedures outlined in this document have been optimized for the detection of protein on Nitrocellulose, PVDF and Nylon membranes. As shown in Table 1, the kinetics of light emission differ significantly between the various types of membranes. All membranes may be exposed to film immediately following a short incubation in Chemiluminescent Substrate Solution. Exposure times vary greatly depending on membrane type. Exposures range from 30 seconds to 15 minutes for PVDF, 5 to 15 minutes for nylon, and 10 to 45 minutes for nitrocellulose. Light emission will persist as long as substrate is available to the enzyme. Table 1: Kinetics of Light Emission of Starlight on Various Membranes Nitrocellulose* x-ray Exposure Maximum Light Levels PVDF* Nylon minutes 1 hour 30 seconds - 15 minutes 2 hours 5-15 minutes 4-6 hours * Treatment with Opti-Membrane Following treatment with Opti-Membrane, the signal intensity is approximately 100 times as bright on PVDF as it is on nitrocellulose. Thus, PVDF requires much shorter film exposures. Opti-Membrane should not be used on Nylon membranes, and although not required with PVDF, faster exposure times will occur with use of Opti-Membrane. 4. Blocking Procedures and Antibody Incubations All volume recommendations refer to the processing of a single blot (10 cm x 10 cm). If larger sections of membrane or multiple blots are being processed simultaneously, the volumes must be increased accordingly. All incubations and washes should be performed at room temperature with constant agitation. It is critical when working with PVDF membranes to keep the membrane wet at all times during the procedure. The complete removal of methanol from the membrane is also essential in attaining uniform low background signal. Pre-wet the membrane by immersing in 100% methanol. Slowly add deionized water while shaking until the methanol concentration is reduced to approximately 20% of the total volume. Do not allow the membrane to dry, during the following procedure. If the membrane does dry repeat this procedure. Place the membrane in transfer buffer for 15 minutes while shaking. We recommend 20 mm Tris, 60 mm Glycine, 20% methanol. Transfer buffers and transfer conditions should be optimized for the particular proteins being studied. See Section 8.A for solution recipes. Note: Membranes must be handled carefully. Mishandled membranes exhibit high non-specific background signals. NEVER TOUCH MEMBRANES WITH UNGLOVED HANDS. 1. Rinse the membrane with PBS and then perform a final wash with PBS for 5 minutes (50 ml). Note: Membranes may be stored in 1X PBS for up to three days at 4 C. 2. Incubate in Blocking Buffer for 30 to 60 minutes (use 50 ml). 3. Dilute the primary antibody in Blocking Buffer. Incubate with primary antibody for 30 to 60 minutes (10 ml). 4. Wash for a minimum of 2X 5 minutes in Blocking Buffer (50 ml) for PVDF. In the case of Nylon and Nitrocellulose, wash for a minimum of 2X 5 minutes in Wash Buffer (50 ml/wash). 5. Dilute secondary antibody-alkaline phosphatase conjugate 1:5000 in Blocking Buffer. (1 ul of conjugate diluted into 5 ml of Blocking Buffer). Incubate with diluted conjugate solution for 30 to 60 minutes. 6. For nylon and nitrocellulose, wash 3X 5 minutes in Wash Buffer (50 ml per wash). For PVDF wash 3X 5 minutes in Blocking Buffer (50 ml per wash). 7. Proceed to Chemiluminescent Detection. 5. Procedure for Chemiluminescent Detection of Proteins See Section 8.B for solution recipes. 1. Wash 2X 2 minutes with 1X Assay Buffer (50 ml per wash). 2. For Nitrocellulose and PVDF membranes incubate for 5 minutes in Chemiluminescent Substrate Solution containing

3 Opti-Membrane. (150 ul Opti-Membrane in 3 ml StarLight ready-to-use solution). Use 3 ml of Substrate Solution per 100 cm 2. The use of Opti-Membrane is optional with PVDF. However, exposure times will be shorter with the use of Opti-Membrane. Note: A separate container should be reserved for incubations with Opti-Membrane as this material is difficult to remove and may cause problems if allowed to contaminate the membrane during other steps. Alternatively, a sealed plastic bag can be used. For Nylon membranes, incubate for 5 minutes in Chemiluminescent Substrate Solution. Use 5 ml of Substrate Solution per 100 cm Drain excess Substrate Solution from the membrane and place it in a Development Folder (after removing the anti-static sheet). Alternatively, the membrane may be wrapped in Saran wrap. Expose to film (See Section 6 below). 6. Film Exposure and Development Membranes wrapped in plastic wrap may be imaged by placing them in direct contact with standard Kodak XAR x-ray film or on Polaroid Instant Photographic Black and White film. Initial exposures of 5 to 30 minutes are recommended to assess the optimal exposure time for your application. Note that exposure times with PVDF membranes are much shorter than with nitrocellulose membranes. Imaging of small blots (up to 7.3 cm X 9.5 cm) may be performed with contact exposures on instant film using an instant film format luminometer with Polaroid Type 612 Instant B/W film ASA 20,000 or Polaroid Type 667 film, ASA The recommended processing time for instant film in this application is 45 seconds. 7. Troubleshooting Guide Since the StarLight substrate provides extremely sensitive detection of alkaline phosphatase activity, it is recommended that only ultrapure water and other reagents which are free of alkaline phosphatase and bacterial contamination be used. The above protocols have been optimized using the alkaline phosphatase conjugated antibodies, Aurora Blocking Reagent, Opti-Membrane and Assay Buffer supplied in the kit. If other enzyme conjugates or blocking reagents are used, results may vary. If the expected sensitivity is not attained, you should try the following: 1. Optimize gel electrophoresis conditions, transfer buffer and electrotransfer conditions. 2. To detect lower concentrations of proteins, lengthen the film exposure time as much as possible or until non-specific background signal obscures the image. 3. Increase the concentrations of primary antibody or alkaline phosphatase conjugate in order to increase signal. This may, however, contribute to increased non-specific binding and subsequent higher background interference. 4. Increase incubation times with primary antibody and alkaline phosphatase conjugate. If the non-specific background is too high (e.g., the film appears overexposed or the image is uneven or spotty) you should try the following: 1. Splotchy images may result from bacterial contamination of the membrane or incomplete or uneven removal of methanol from the membrane prior to blocking. Make sure that all reagents and buffers are kept free of contamination prior to use and that the membrane is clean and free of fingerprints. 2. In non-specific binding of the antibodies is high, filter the antibody. Dilute the antibody to 2X the final working dilution in Wash Buffer and filter through a 0.45 micron filter, then further dilute 1:1 with Blocking Buffer. 3. If the background signal appears to obscure the specific signal, decrease the exposure time until appropriate resolution is achieved. 4. If decreased exposure time does not improve resolution, increase to 15 minutes the wash steps which follow the incubation with the primary antibody and alkaline phosphatase conjugated secondary antibody. Concurrently increasing the concentration of the Tween-20 component of the Wash Buffer to 0.3% to 0.5% may also help. 5. Blocking non-specific binding sites on the membrane may be improved by incubating the membrane in Blocking Buffer overnight at 4 C. 6. It may help to increase the dilution of secondary antibody-alkaline phosphatase conjugate to 1:20,000 to 1:50,000 to reduce non-specific signal. 7. If Opti-Membrane has been used with PVDF membrane and a high background is obtained, omit the Opti-Membrane reagents. 8. Preparation of Solutions All solutions should be made with filtered, deionized water. It is recommended that all solutions be made fresh prior to use in order to prevent possible contamination from bacterial alkaline phosphatase. Solutions may be stored for short periods of time with the addition of 0.02% sodium azide and storage at 4 C.

4 8.A Preparation of Solutions for Membrane Blocking 10X Phosphate Buffered Saline (PBS) To Make 1000 ml, Use: 0.58 M Na2HPO g of Na2HPO M NaH2PO4-H2O 23.5 g of NaH2PO4-H2O 0.68 M NaCl 40.0 g of NaCl Add deionized water to 1000 ml. Note: A 1 X PBS solution should have a ph of 7.3 to ml is sufficient to process approximately 20 blots. Alternatively pre-made solutions can be purchased from MP: Catalog Number Description Size X PBS without calcium, magnesium 100 ml 500 ml PBS tablets without calcium, magnesium (1 tablet = 100 ml) 100 tab 200 tab 500 tab Wash Buffer To Make 300 ml, Use: 1X PBS 30 ml of 10X PBS 0.1% Tween ml of Tween-20 Add deionized water to 300 ml. For Nitrocellulose and Nylon: Prepare 300 ml per 100 cm 2 blot. For PVDF: Prepare 150 ml per 100 cm 2 blot. Blocking Buffer For all membranes except positively charged Nylon* To Make 100 ml, Use: 0.2% Aurora Blocking Reagent 0.2 g of Aurora Blocking Reagent 1X PBS 10 ml of 10X PBS 0.1% Tween ml of Tween-20 Add 10 ml of 10X PBS to 90 ml of water. Add Aurora Blocking Reagent, microwave for 40 seconds, then stir, or heat on a hot plate. DO NOT BOIL. Add Tween-20 after the solution has cooled. The solution will remain slightly opaque. Cool to room temperature before use. *For positively charged Nylon: Use 3% Aurora Blocking Reagent (3.0 g/100 ml) in 1X PBS with 0.1% Tween-20. For Nitrocellulose and Nylon: Prepare 100 ml per 100 cm 2 For PVDF: Prepare 350 ml per 100 cm 2 For Nylon+: Prepare 150 ml per 100 cm 2 8.B Preparation of Solutions for Chemiluminescent Detection Assay Buffer The kit is supplied with 10X Assay Buffer: 200 mm Tris-HCl, ph 9.8, 10 mm MgCl2. Dilute 1:10 with deionized water. Chemiluminescent Substrate Solution The chemiluminescent substrate, StarLight, is supplied as a ready-to-use solution.

5 8.C Solutions for Membrane Stripping and Reprobing Stripping Buffers 1. b-mercaptoethanol/sds Stripping Buffer To Make 100 ml, Use: 62.5 mm Tris-HCl, ph ml of Tris-HCl 2% Sodium Dodecyl Sulfate (SDS) 1 g of SDS 100 mm b-mercaptoethanol 0.34 ml of b-mercaptoethanol 2. Glycine Stripping Buffer To Make 300 ml, Use: 0.2 M Glycine, ph 2.2* 4.5 g of Glycine 0.1% Sodium Dodecyl Sulfate (SDS) 0.3 g of SDS 1.0% Tween-20 3 ml of Tween-20 * Adjust ph to 2.2 with HCl Wash Buffer (see Section 8.A) Assay Buffer (see Section 8.B) 9. DOT/SLOT Blot Comments For dot blotting or slot blotting of proteins on nitrocellulose, dilute proteins in a Tris/Glycine/Methanol transfer buffer. To prepare this buffer, use 20 mm Tris, 60 mm Glycine, 20% methanol. Air dry the membrane before proceeding with the Blocking Buffer incubation. For slot blotting proteins onto PVDF, wet the membrane in 100% methanol, then equilibrate in Tris/Glycine/methanol transfer buffer. Dilute the proteins in this buffer and apply to the membrane. If the membrane dries out during blotting, rewet in 100% methanol and transfer to Tris/Glycine/methanol buffer before blocking. 10.Stripping and Reprobing Procedure for removal of detection antibodies and chemiluminescent substrate (all steps are performed at room temperature): See Section 8.C for solution recipes. Solutions required in the following procedures are numbered as in Section To strip detection antibodies, wash the membrane for 60 minutes with Stripping Buffer. 2. Prior to incubation with new antibody, wash the blot 3X 5 minutes with Wash Buffer. 3. Follow the Aurora Western Blot protocol, beginning with the incubation in Blocking Buffer. Note: If you wish to verify the complete removal of the detection antibodies from the membrane after performing Steps 1 and 2, incubate the blot 2X 5 minutes in Assay Buffer, 5 minutes in Chemiluminescent Substrate Solution, and then expose the blot to film as performed in Section 6. Complete removal of detection antibodies may be confirmed by the absence of an image on film. To reprobe begin at Step 2 above. The results obtained using this protocol may vary depending on the membrane, its interaction with proteins, and the nature of the primary antibody. References: Towbin, H., Staehelin, T. and Gordon, J., "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications." Proc. Natl. Acad. Sci., (USA), v. 76: (1979). Gershoni, J.M. and Palade, G.E., "Review: Protein blotting: principles and applications," Anal. Biochem., v. 131, 1-15 (1983). Bronstein, I., Edwards, B. and Voyta, J.C., "1,2-Dioxetanes: Novel chemiluminescent enzyme substrates." Applications to Immunoassays," J. of Biolumin. Chemilumin., v. 4, (1989). Gillespie, P.G. and Hudspeth, A.J., "Chemiluminescence detection of proteins from Single Cells," Proc. Natl. Acad. Sci. (USA), v. 88, (1991). Warranty MP warrants its products to the original purchaser against defects in materials and workmanship under normal use and application. MP's sole obligation under this warranty shall be to replace defective products.

6 All products are supplied FOR RESEARCH USE ONLY and are not for resale. Commercialization of products using these components requires an express license under applicable patents and intellectual property from MP. Our preparations are intended exclusively for in vitro use only. They are not for diagnostic or therapeutic use in humans or animals. Those preparations with known toxicity are sent with an information sheet which describes, to our knowledge, the potential dangers in handling. The absence of a toxicity warning with one of our products does not, however, preclude a possible health hazard. With all of our products, due care should be exercised to prevent human contact and ingestion. All preparations should be handled by trained personnel only. This warranty is in lieu of all other warranties, express or implied, including the warranties of merchantability and fitness for a particular purpose. In no case shall MP be liable to incidental or consequential damages. Availability: Catalog Number Description Size Aurora Western Blot Kit - Human 10 blots Aurora Western Blot Kit - Mouse 10 blots Aurora Western Blot Kit - Rabbit 10 blots Aurora Western Blot Kit - Rat 10 blots