INSTRUCTIONS Pierce In-Gel Chemiluminescent Detection Kit for Biotinylated Antibody Probes 33610 Number Description 33510 Pierce In-Gel Chemiluminescent Detection Kit for Biotinylated Antibody Probes, sufficient reagents for 10 mini-gels (1,000 cm 2 ) 1338.3 Kit Contents: Streptavidin-Horseradish Peroxidase (Streptavidin-HRP), 1 mg, lyophilized Dilution Buffer (10X), 50 ml BupH Phosphate Buffered Saline Packs, 17 packs, each pack makes 500 ml of 0.1 M phosphate, 0.15 M sodium chloride; ph 7.2 Luminol Enhancer, 55 ml Stable Peroxide, 55 ml Surfact-Amps 20, 6 10 ml vials, 10% Tween -20 Solution Pre-Cut Cellophane, 10 sheets Storage: Upon receipt store kit at 4 C. Product is shipped at ambient temperature. The Streptavidin- HRP is stable for 6 months at 4 C after reconstitution. Introduction The Pierce In-Gel Chemiluminescent Detection Kit for Biotinylated Antibody Probes allows antigen detection directly within a polyacrylamide gel. Historically direct probing of polyacrylamide gels has been problematic and Western blotting has remained necessary. However, with an optimized pretreatment step and the introduction of an extremely sensitive chemiluminescent substrate, it is now possible to detect antigens within the gel. Although only a limited amount of antibody enters the gel, this amount is sufficient for detection by a sensitive chemiluminescent substrate. Because gels are threedimensional, protein interactions within the gel result in complexes exhibiting specific and intense bands. Furthermore, gel development time is much shorter than is required for a Western blot. Traditional Western blotting involves protein separation in a slab gel followed by transfer of the proteins to a membrane. The membrane is incubated with a specific antibody that is then detected via a reporter system. 1,2 Inherent in each of the Western blotting steps are factors that affect the overall transfer efficiency. For example, the electroelution efficiency of protein depends on the electric current used, the gel pore size and the proteins molecular weights and net charge. Transfer conditions suited for one protein may not be optimal for another because transfer of a smaller protein is more efficient than that of a larger one. The membrane pore size can also affect the overall immobilization of protein, and diffusion during electrotransfer can affect protein band widths on the membrane. 3 Western blotting techniques have other known limitations. In addition to sample preparation and electrophoresis, blotting conditions can destroy relevant epitopes. Protein renaturation is often inefficient and/or incomplete and because specific antibodies are frequently raised to proteins in their native conformations, the binding of antibodies to blotted proteins is unpredictable. 3 Studies also indicate that antigens can be lost from membranes during immunoprocessing. 4 These factors suggest that patterns obtained on a Western blot may not be a true representation of the sample separated by electrophoresis. In addition, some proteins do not transfer well to membranes and others bind selectively to different membranes. 5 In-gel detection overcomes the limitations of traditional protein detection with an effective method to directly detect proteins in a polyacrylamide gel. Without the need to transfer proteins, this technique enables results to be obtained in four hours after gel electrophoresis.
Important Product Information This product is designed for detecting antigens using biotinylated antibodies as probes. This kit requires either a purchased biotinylated antibody or an antibody biotinylated in the lab (see Additional Information section). The following pre-cast gel brands and types work well with the in-gel method: Novex, FMC, BioWhittaker and Bio- Rad Criterion brand Bis-Tris, Tris-Glycine, Tricine and Tris-Acetate gels (both native and denaturing). Pre-cast Bio-Rad, igel and Zaxis gels do not perform well with the in-gel method. Sensitivity with these gel types is 25X less, and they may require individual optimization. Homemade gels can be used (but see modifications to default procedure in Material Preparation section). For optimal results use gels that are 0.75-1.5 mm thick. Gradient gels of 3-8%, 4-12%, 8-18%, 4-20% and 10-20%, as well as homogeneous gels (8-16%) work well. Proteins of MW 20,000-160,000 have been detected successfully with the in-gel method. Use a clean knife or plastic spatula to manipulate the gel. To avoid background, do not touch gel with your hands. Use a platform shaker for all incubation steps. Keep gel submerged during all incubation and wash steps. Note: Optimization of the antigen, biotinylated antibody and Streptavidin-HRP dilutions is necessary to achieve the best results for your specific application. Procedure Summary 1. Pre-treat gel for 15 minutes in 50 ml of 50% isopropyl alcohol, or, when using Precise Protein Gels, 40% methanol. 2. Wash gel in 100 ml ultrapure water for 15 minutes. 3. Incubate gel in 20 ml biotinylated antibody solution (final concentration 0.1-2.0 µg/ml) for 1 hour. Wash gel in PBS-T. 4. Incubate gel in 20 ml Streptavidin-HRP (1:5,000-1:20,000 dilution of prepared stock) for 1 hour. Wash gel in PBS-T. 5. Incubate gel in 20 ml Substrate Working Solution (1:1 solution of each component) for 5 minutes. 6. Rinse gel in ultrapure water for 15 seconds. Sandwich gel between cellophane sheets and expose to film or CCD camera. Material Preparation Homemade polyacrylamide gels Purified proteins Lysates Streptavidin-HRP For homemade gels make the following modifications to the default procedure: Precoat glass plates of gel-casting mold with a siliconizing agent. Use AquaSil Siliconizing Fluid (Product No. 42799) by add 5 ml of the solution to 95 ml of distilled water. Dip plates in the diluted solution, then dry plates overnight at room temperature or 1-2 hours at 100 C. Rinse dry plates with distilled water for immediate use. Plates do not require retreatment for several uses. If using a different siliconizing agent, follow the manufacturer s instructions for plate preparation. Eliminate SDS from bis/acrylamide polymerization mix (there is sufficient SDS in sample loading buffer to ensure correct electrophoresis of sample). In step 3 of procedure, pretreat gels using 50% methanol rather than isopropyl alcohol. If homemade gels are cast in disposable plastic gel cassettes, surface treatment of the cassette plastic generally is not necessary. Eliminate SDS from the bis/acrylamide polymerization mix. No alteration to step 3 is necessary (isopropyl alcohol can be used). Dilute purified proteins and combine with SDS-PAGE sample buffer so that 10 µl of the diluted sample contains 1-10 ng of protein. Heat for 5 minutes at 95 C, and apply sample to well. Combine lysate with SDS-PAGE sample buffer so that 10 µl of sample contains 1-10 ng of target protein. (Depending on the lysate sample and protein to be detected, between 1:2 and 1:100 dilution of the lysate will be appropriate. Heat for 5 minutes at 95 C, and apply to well. Reconstitute the 1 mg Streptavidin-HRP with 1 ml of PBS. Reserve some PBS without the Tween-20 for this reconstitution step. Product is stable for 6 months at 4 C after reconstitution. 2
PBS with 0.05% Tween-20 (PBS-T) Dilution Buffer Prepare 2 L of PBS-T by combining four packets of BupH Phosphate Buffered Saline and one ampule of Surfact-Amps 20. Approximately 650-700 ml of buffer is required for one gel. Add 4 ml of Dilution Buffer (10X) to 36 ml of PBS-T. 50% isopropyl alcohol Add 25 ml of 100% isopropyl alcohol (not included in kit) to 25 ml of ultrapure water and mix. Procedure for In-Gel Detection Notes: Negative control: If endogenous biotin is expected in the sample or if the Streptavidin-HRP binds nonspecifically to protein bands in the sample, then perform the following steps: a. Perform SDS-PAGE on two identical gels and use one gel as a negative control. b. Follow with the Procedure for In-Gel Detection until step 6, at which point incubate the negative control gel with the Dilution Buffer alone (i.e., no biotinylated primary antibody) while incubating the test gel with the biotinylated antibody. c. Complete the Procedure for In-Gel Detection with both gels. Gel handling: Use a clean knife or plastic spatula to manipulate the gel. Do not touch gel with hands, which can produce high background. Use a platform shaker for all incubation steps. The gel must be completely submerged throughout all incubation and wash steps. Use the Incubation Colander (Product No. 33499, see Additional Information section) or other suitable container for the incubation and wash steps. Use a second clean container for substrate incubation step. 1. After electrophoresis, remove the top plate of gel cassette. Use a knife to cut off and discard the top (stacking) portion of gel, approximately 0.5-1 cm from the bottom of the wells (see Figure 1). Note: Removing the top of gel reduces background during film exposure. If the protein of interest is of high M.W., cut only a small amount from the top of the gel, or remove this part of the gel after the first exposure if it causes high background. Note: Notching one top corner of gel (see Figure 1) can assist in orientating the results when detectable molecular weight markers are not used. 8 cm 10 cm Figure 1: Gel cutting and orientation. 2. Carefully transfer gel to clean polypropylene tray (or Incubation Colander, see Related Products). 3. Pre-treat gel for 15 minutes in 50 ml of 50% isopropyl alcohol, or, when using Precise Protein Gels, 40% methanol. Gently shake gel in this solution for 15 minutes. 4. Replace alcohol with 100 ml of ultrapure water. Wash gel with gentle shaking for 15 minutes. This can be a stopping point. Gel can be left in ultrapure water or 10% isopropyl alcohol overnight at 4 C. Note: Between steps, lift out the colander with gel and completely drain all liquid from the tray. Remove excess liquid from the colander by tapping and/or blotting the bottom onto clean paper towel. Note: During the wash step, prevent gel curling by gently flattening the gel with a gel knife or spatula or flipping the gel over. The gel must be completely submerged in water to remove excess alcohol from the gel surface. 5. Dilute biotinylated antibody to a final concentration of 0.1-2.0 µg/ml in 20 ml of Dilution Buffer. For example, to make 0.1 µg/ml biotinylated antibody solution, add 20 µl of antibody stock at 0.1 mg/ml to 20 ml of the Dilution Buffer (see the Troubleshooting section for alternative dilution buffers). Note: Many biotinylated primary antibodies are commercially available; however, if a biotinylated antibody necessary for your research is not available, see the Additional Information section for a list of biotin labeling reagents. Note: If the primary antibody is valuable, prepare only 10 ml of primary antibody solution and perform the incubation step in a smaller container (e.g., a petri dish). If needed, excise unused portions of the gel so that it fits the smaller container. All wash steps may be performed in a regular tray. 3
6. Decant wash water from the gel container. Add diluted antibody solution and incubate gel for 1 hour at room temperature (RT). Make sure gel floats freely in the solution. This can be a stopping point. Gel in covered colander can be left to incubate in primary antibody solution overnight at 4 C (shaking is not required). 7. Wash gel 3 10 minutes each with 100 ml PBS-T. Gel must float freely in solution and be shaken gently. 8. Dilute the reconstituted 1 mg/ml Streptavidin-HRP 1:5,000-1:20,000 in Dilution Buffer. For example, to prepare 1:10,000 dilution, first prepare a 1:10 dilution by adding 5 µl of the reconstituted Streptavidin-HRP (1 mg/ml stock) to 45 µl of Dilution Buffer in a microcentrifuge tube and mix. Then add 10 µl of the 1:10 diluted Streptavidin-HRP to 10 ml of Dilution Buffer. 9. Dilute the reconstituted 0.1 mg/ml Streptavidin-HRP 1:500-1:2,000 in Dilution Buffer. To make 1:500 dilution of the antibody stock, add 40 µl of the reconstituted Streptavidin-HRP (0.1 mg/ml) to 20 ml of Dilution Buffer. 10. Decant wash buffer and add diluted Streptavidin-HRP solution. Incubate gel for 1 hour at RT. 11. Wash the gel 3 10 minutes each with 100 ml PBS-T. Gel must float freely in solution and be shaken gently. 12. Prepare the Substrate Working Solution just before use by mixing 5 ml of the Stable Peroxide with 5 ml of the Luminol Enhancer. Note: Exposure to the sun or any other intense light can harm the Working Solution. For best results keep the Working Solution in an amber bottle and avoid prolonged exposure to any intense light. Typical laboratory lighting will not harm the Working Solution. 13. Add 10 ml of Substrate Working Solution to a clean tray. 14. Transfer the gel to the tray containing Substrate Working Solution. Use only the bottom tray of the colander device for substrate development. 15. Incubate gel in 10 ml of the Substrate Working Solution for 5 minutes at RT with gentle shaking. Both sides of gel must be exposed to the substrate. 16. Decant Substrate Working Solution. Add 50 ml of ultrapure water, wash for 15 seconds and decant water. Do not wash for more than 15 seconds or poor signal will result. 17. Place gel between two cellophane sheets. Remove excess liquid from around gel by wicking it away with a strip of filter paper or paper towel; do not allow paper to contact the gel surface. Gently smooth out air bubbles from gel sandwich. 18. Expose the gel sandwich to film for different lengths of time and develop the film, or expose gel to a CCD camera. The chemiluminescent signal can be detected for up to 2 hours following the substrate incubation step. Note: Try exposing film for 1 minute. If no bands are detectable, then increase exposure time to 5 minutes or longer. If the 1 minute exposure produces overly dark bands and background, decrease exposure time to 5-30 seconds. 4
Stripping and Reprobing the Gel A. Additional Materials Required Stripping Buffer: Restore Western Blot Stripping Buffer (Product No. 21059, See Related Pierce Products) Oven set to 37 C B. Procedure 1. Carefully open cellophane sheets that sandwich gel and add a few ml of ultrapure water to loosen gel from sheets. 2. Remove gel from cellophane with a gel knife and place it in a tray with 20 ml of Stripping Buffer. 3. Cover tray with plastic wrap to prevent evaporation, and incubate gel for 30-60 minutes at 37 C with gentle shaking. Make sure gel floats freely in the solution. 4. Decant Stripping Buffer and wash gel 3 10 minutes with 50 ml of PBS-T with gentle shaking. This can be a stopping point. Gel can be left in ultrapure water overnight at 4 C. 5. Ensure that gel has been completely stripped by repeating Steps 11-15 of the In-Gel Detection Procedure. Note: If bands are still visible, repeat Steps 1-5, incubating gel in Stripping Buffer for an additional 15 minutes at 37 C. If no bands are detectable following exposure, assume that the gel has been stripped completely and proceed to Step 6. 6. Carefully open cellophane sheets that sandwich gel and add a few ml of ultrapure water to loosen gel from sheets. 7. Remove gel from cellophane sheets, place it in a tray and wash it 3 5 minutes with 50 ml PBS-T with gentle shaking. Make sure gel floats freely in the solution. 8. Re-probe gel by returning to the In-Gel Detection Procedure beginning with Step 5. Staining for Total Protein after In-Gel Detection A. Additional Materials Required Stain: GelCode Blue Stain Reagent (Product No. 24590, see Related Products) Destain Solution: 200-500 ml of 55% ultrapure water/5% acetic acid/40% methanol (v/v/v) B. Procedure 1. Carefully open cellophane sheets that sandwich the gel and add a few ml of ultrapure water to loosen gel from sheets. 2. Remove gel from cellophane sheets, place it in a tray and rinse it 3 5 minutes with 50 ml ultrapure water with gentle shaking. Leave the gel immersed in the water wash for at least one hour (overnight is preferable). Note: Insufficient washing will cause high background mainly at the top of the gel 3. Decant rinse water and add 50 ml GelCode Blue Stain Reagent. Incubate for 1 hour at room temperature, making sure gel floats freely and remains covered with stain solution. 4. Decant Stain and rinse gel 2 5 minutes with 50 ml ultrapure water with gentle shaking. 5. Destain gel with 50 ml aliquots of Destain Solution with gentle shaking. Change solution periodically until gel and the solution are clear. 6. Store stained gel in ultrapure water until ready for gel documentation or preservation. 5
Troubleshooting Problem Possible Cause Solution High background at Did not cut off enough of stacking gel Cut 0.5-1 cm from the bottom of the comb the top of gel High background Biotinylated primary antibody Further dilute biotinylated primary antibody (e.g., concentration too high (e.g., 200 ng/ml) 100 ng/ml) Streptavidin-HRP concentration too high (e.g., 1:5,000 dilution or 200 ng/ml) Reduce Streptavidin-HRP concentration (e.g., Dilute 1:20,000 or 50 ng/ml) Washes inadequate and/or gel stuck to tray during wash steps Increase the length (e.g., 3 20 min 50 ml/wash) or frequency (e.g., 6 5 min 50 ml/wash) of washes. Make sure the gel floats freely in wash solution Substrate incubation too long and/or water wash was omitted Film exposure too long Reduce the substrate incubation and/or follow with water rinse Reduce the film exposure (e.g., from 1 minute to 30 seconds or 15 seconds) Correct film overexposure with Erase-It Background Eliminator (see Related Pierce Products) Low signal Streptavidin-HRP diluted too much Try two- to five-fold higher concentration Biotinylated primary antibody diluted too much Try two- to five-fold higher biotinylated primary antibody concentration. Alter biotinylated antibody concentration only if adjusting Streptavidin-HRP concentration has no effect Diffuse bands Residual isopropyl alcohol remaining on Make sure gel does not stick to tray, is totally submerged gel surface following pretreatment step and floats freely during water wash Gel electrophoresed too quickly Decrease voltage during electrophoresis Gel was old (i.e., expired) Use gels that have not expired. Expired gels affect In-Gel Detection more significantly than Western blotting. Fuzzy bands Protein degradation Prepare new protein samples Nonspecific bands Dilution Buffer incompatible (e.g., primary antibody cross-reacts with protein in Dilution Buffer) Try one of the following alternative Dilution Buffers: 1 mg/ml Goat Gamma Globulin (Product No. 31869), 1X SuperBlock Blocking Buffer (Product No. 37515) No bands Protein did not migrate from stacker Use lower percent polyacrylamide gel (i.e., percent gel was too high) Biotinylated antibody does not recognize Ensure by other methods that biotinylated antibody Additional Information target protein Streptavidin-HRP does not bind the antibody because it is not in fact biotinylated recognizes the target protein Ensure by other methods that the primary antibody was successfully biotinylated A. To biotinylate antibodies in the laboratory, use one of the following reagents: Sulfo-NHS-LC-Biotin (Product No. 21335): This reagent biotinylates the IgG through the primary amine groups on the antibody and is most commonly used to biotinylate antibodies. The procedure for biotinylation is simple and can be performed while the gel is being electrophoresed. This reagent is also available in a kit format (Product No. 21420). Biotin-BMCC (Product No. 21900): This reagent biotinylates the IgG through the sulfhydryl groups that are produced by reduction of the antibody with 2-Mercaptoethylamine HCl (Product No. 20408) or introduced through the primary amine groups of the antibody by N-Succinimidyl-S-acetylthioacetate (SATA, Product No. 26102) or 2-Iminothiolane HCl (Traut s Reagent, Product No. 26101) Biotin-LC-Hydrazide (Product No. 21340): This reagent biotinylates the IgG through the carbohydrate moieties on the Fc region. 6
B. The Incubation Colander device Using the gel colander device (Product No. 33499) for gel incubation and wash steps reduces gel handling and, therefore, results in less chance of gel tearing. The device is supplied with two bottom trays and one colander (Figure 2). B C A Figure 2. Schematic of the Incubation Colander device. The schematic is labeled as follows: A and C are the bottom trays and B is the colander. Related Products 33500 Pierce In-Gel Chemiluminescent Detection Kit Rabbit 33505 Pierce In-Gel Chemiluminescent Detection Kit Mouse 33550 Pierce Chemiluminescent Substrate*, 110 ml, sufficient to detect horseradish peroxidase- conjugated probes on 10 mini-gels (8 cm 10 cm) 33499 Incubation Colander, 1 unit 34090 CL-XPosure Film, 5 7 sheets, 100 sheets/pkg 21059 Restore Western Blot Stripping Buffer, 500 ml, sufficient to strip 25 mini-gels or Western blots (8 cm 10 cm) 21065 Pierce Background Eliminator, 200 ml, sufficient to correct overexposure of at least 10 films (5 7 ) 24590 GelCode Blue Stain Reagent, 500 ml, sufficient to stain 25 mini-gels (8 cm 10 cm) References 1. Towbin, H., et al. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci U.S.A. 76:4350-4. 2. Renart, J., et al. (1979). Transfer of proteins from gels to diazobenzyloxymethyl paper and detection with antisera. A method for studying antibody specificity and antigen structure. Proc Natl Acad Sci U.S.A. 76:3116-20. 3. Lin, W. and Kasamatsu, H. (1983). On electrotransfer of polypeptides from gels to nitrocellulose membranes. Anal Biochem 128:302-11. 4. DenHollander, N. and Befus, D. (1989). Loss of antigens from immunoblotting membranes. J Immunol Meth 122:129-35. 5. Reddy, V.M. and Kumar, B. (2000). Interactions of Mycobacterium avium complex with human respiratory epithelial cells. J.I.D 181:1189-93. Tween is a registered trademark of ICI Americas. This product ( Product ) is warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale, as set forth in the Product documentation, specifications and/or accompanying package inserts ( Documentation ) and to be free from defects in material and workmanship. Unless otherwise expressly authorized in writing, Products are supplied for research use only. No claim of suitability for use in applications regulated by FDA is made. The warranty provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the Documentation, this warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. This warranty does not extend to anyone other than the original purchaser of the Product ( Buyer ). No other warranties, express or implied, are granted, including without limitation, implied warranties of merchantability, fitness for any particular purpose, or non infringement. Buyer s exclusive remedy for non-conforming Products during the warranty period is limited to replacement of or refund for the non-conforming Product(s). There is no obligation to replace Products as the result of (i) accident, disaster or event of force majeure, (ii) misuse, fault or negligence of or by Buyer, (iii) use of the Products in a manner for which they were not designed, or (iv) improper storage and handling of the Products. Current versions of product instructions are available at www.thermo.com/pierce. For a faxed copy, call 800-874-3723 or contact your local distributor. 2007 Thermo Fisher Scientific Inc. All rights reserved. Unless otherwise indicated, all trademarks are property of Thermo Fisher Scientific Inc. and its subsidiaries. Printed in the USA. 7