For best results, optimization of the primary and secondary antibody concentrations is recommended.

INSTRUCTIONS UnBlot Chemiluminescent Substrate 3747 N. Meridian Road P.O. Box 117 Rockford, IL 61105 33550 1289w Product Description Number Description 33550 UnBlot Chemiluminescent Substrate Kit contents: UnBlot Luminol Enhancer, 55 ml UnBlot Stable Peroxide, 55 ml Notes: Storage: Store the kit at 4 o C. The kit includes enough reagents to cover 1,000 cm 2 (10 mini-gels). Note: UnBlot Chemiluminescent Substrate is used with the UnBlot In-Gel Chemiluminescent Detection Kits. The UnBlot In-Gel Chemiluminescent Detection Kit has been tested successfully with Novex, FMC, BioWhittaker and Bio-Rad Criterion brand Bis-Tris, Tris-Glycine and Tricine gels. UnBlot In-Gel Chemiluminescent Detection Kit does not work well with regular Bio-Rad gels, igel and Zaxis gels. Studies showed 25X lower sensitivity and require individual optimization. Homemade gels may require further optimization. The recommended gel thickness for use with this kit is 0.75-1.5 mm. Gradient gels of 3-8%, 8-18%, 4-20% and 10-20%, as well as nongradient gels (8-16%) work well with the UnBlot In-Gel Chemiluminescent Detection Kit. Proteins of M.W. 20,000-160,000 have been used successfully with the UnBlot In-Gel Chemiluminescent Detection Kit. For best results, optimization of the primary and secondary antibody concentrations is recommended. 1

Introduction Polyacrylamide gel electrophoresis is commonly used to separate protein samples. To determine the complexity of the samples, the gels can be stained for proteins. However, detection of specific interactions was often necessary and was performed directly in the gel. 1,2,3 The procedures took several days as they entailed lengthy fixing, incubation and wash steps. 4 Detection was performed either with corresponding peroxidase-labeled antibody and 3,3-diaminobenzidine or with radiolabeled lectins or antibodies. To improve signal-to-noise ratios and to shorten the protocol, the detection within the gel was replaced with Western blotting techniques. 5,6 The strategy for performing Western blotting is to separate the proteins in a slab gel and transfer the pattern from a 3-dimensional gel to a 2-dimensional membrane matrix where it is immobilized. The membrane is incubated with a specific antibody, which in turn is detected with a reporter. Reporters are radiolabeled, fluorescent or enzyme-labeled molecules. 7 Inherent to each of the Western blotting steps are factors that affect the overall transfer efficiency of this method. For instance, the electroelution of polypeptides depends on the electric current used, the pore size of the polyacrylamide gels, the molecular weights and the net charge of the polypeptides. Transfer conditions that are suited for one polypeptide may not be optimal for another as the rate of elution of each polypeptide is dependent on its molecular weight. The transfer of a smaller polypeptide is more efficient than that of a larger one. The pore size of the membrane may also affect the overall immobilization. Diffusion during electrotransfer may affect the band widths of the polypeptides immobilized on nitrocellulose. 8 Western blotting techniques are also known to have certain limitations because, in addition to sample preparation and electrophoresis, blotting conditions can destroy the relevant epitopes. 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. 7 Studies have also shown that antigens can be lost from membranes during immunoprocessing. 9 These factors suggest that the pattern obtained on a Western blot may not be a true representation of the sample separated by electrophoresis. There are also some proteins such as fibrinogen, which do not transfer well to membranes. To overcome the limitations of Western blotting, Pierce has developed the UnBlot Technique whereby antigens can be detected inside the polyacrylamide gel. Thus far polyacrylamide gels were not used for direct probing because of the general belief that they are impermeable to antibodies and other large proteins. With an optimized pretreatment step and the current introduction of an extremely sensitive chemiluminescent substrate it is possible to detect antigens within the gel using corresponding antibodies. Although only a limited amount of antibody or other protein may enter the polyacrylamide gel, the amount is sufficient to be detected by a sensitive chemiluminescent substrate. Also, because gels are 3-dimensional, interactions within the gels result in complexes. Therefore, specific and intense bands are obtained when interactions occur. Specific antigens have been detected from a separation of a crude sample. The development of the gel is shorter than the time required for developing a Western blot. 2

Make Ready 1. a. Purified proteins: Dilute purified proteins to twice the desired concentration in a buffer such as PBS, then dilute further using 2X SDS-PAGE sample buffer. The final dilution is made so that 10 µl of the diluted sample contains 1-10 ng of protein. Heat for 5 minutes at 95 C, then load the gel. b. Lysates: Dilute lysates from 1:2 to 1:100 in 2X SDS-PAGE sample buffer. Heat for 5 minutes at 95 C, then load 10 µl of the diluted sample to the wells. (20 µl is used for a 10-well gel.) 2. PBS/0.05% Tween -20: For each 2 liters to be prepared, use 4 packets of BupH Phosphate Buffered Saline and 1 ampule of Surfact-Amps -20. The volume required per gel is approximately 650-700 ml. 3. Antibody Dilution Buffer: Add 4 ml of Antibody Dilution Buffer (10X) to 36 ml of PBS/0.05% Tween -20. 4. UnBlot Substrate Working Reagent: Mix 5 ml of the UnBlot Stable Peroxide with 5 ml of the UnBlot Luminol Enhancer. Prepare the working reagent fresh. 5. 50% isopropanol: Add 25 ml of 100% isopropanol to 25 ml of ultrapure water. Mix. Notes: i. Incubations: Use a platform shaker for all of the incubation steps. Gels should float freely in the colander during all incubations. ii. iii. iv. Substrate Development: Transfer the gel into a clean incubation tray containing the UnBlot Substrate Working Reagent when performing the substrate development step. Gel Orientation: Cut one of the top corners of the gels to help with orientation of the gel (M.W. marker does not transfer to the film). Wash and incubation steps: The gel must be completely submerged throughout all incubation and wash steps. The gel colander device (Product No. 33499) should be used for all incubations and wash steps. Use a second clean tray for substrate incubation. Sample Protocol Intact Bio-Rad Criterion gels will not fit in the colander device unless they are cut in half. For intact gels, please use a clean polypropylene container. 1. Following electrophoresis, remove and discard the top plate, allowing the gel to remain on the bottom (slotted) gel cassette. Using the gel knife, cut the gel across the top (approximately 0.5-1 cm from the bottom of the wells) and discard this top strip. Next, cut one top corner of the gel. After removing the second side of the cassette, place the gel in the colander device. 8cm 10 cm Notes: Cutting the top of the gel reduces the background during the film exposure. Cutting one top corner of the gel allows for easy gel orientation during the film exposure because M.W. markers do not transfer to the film (with longer exposures, ghost bands of the M.W. marker may appear on the film). 3

2. PRETREATMENT STEP: Add 50 ml of 50% isopropanol in ultrapure water to the colander device containing the gel and pretreat for 15 minutes with mild agitation. 3. WASHING STEP: Replace the alcohol in the colander device with 100 ml of ultrapure water and wash the gel for 15 minutes with mild agitation. Note: Between steps, drain all the liquid from the colander device. Remove excess liquid by tapping and/or blotting on the paper towel. Note: The gel has tendency to curl up. To eliminate curling, flatten the gel gently with a gel knife or spatula or flip the gel over. The gel needs to be completely submerged in water to remove the excess alcohol from the surface of the gel. This can be a stopping point. The gel can be left in the water overnight at 4 C. 4. PRIMARY ANTIBODY STEP: For rabbit and mouse systems, dilute the primary antibody in Antibody Dilution Buffer* to a final concentration of 0.1-2.0 µg/ml in 20 ml for rabbit, 0.1-2.5 µg/ml in 20 ml for mouse. Lift the colander containing the gel and pour out the water from the colander base. Add the antibody solution to the colander device and incubate for 1 hour at room temperature. (Example of antibody dilution: To make a 0.1 µg/ml antibody solution, add 20 µl of your antibody stock at 0.1 mg/ml to 20 ml of the Antibody Dilution Buffer.) Note: Make sure that the gel is floating freely in the solution. *If you do not have access to Pierce's Antibody Dilution Buffer, substitution of a blocking buffer, such as BSA or Casein, should provide satisfactory results. This can be a stopping point. You can leave the gel in the primary antibody overnight at 4 C (no agitation required). 5. WASH STEP: Wash the gel 3 x 10 minutes with 100 ml PBS/0.05% Tween -20. Note: Make sure that the gel is floating freely in the solution during mild agitation. 6. SECONDARY ANTIBODY STEP: Dilute the goat anti-rabbit (GAR) (1:50,000) or goat anti-mouse (GAM) (1:25,000) horseradish peroxidase-conjugated (HRP) antibody in Antibody Dilution Buffer. Pour off the wash buffer and add GAR or GAM HRP antibody. Incubate the gel for 1 hour at room temperature. 7. WASH STEP: Wash the gel 3 x 10 minutes with 100 ml PBS/0.05% Tween -20. 8. SUBSTRATE STEP: Remove the gel from the colander and transfer directly to the clean tray. Incubate the gel in 10 ml of the UnBlot Substrate Working Reagent for 5 minutes at room temperature with mild agitation. Note: Make sure that both sides of the gel are exposed to the substrate. 9. RINSE STEP: Pour off the UnBlot Substrate and replace with 50 ml of ultrapure water. Wash the gel for 15 seconds and then pour the water off the tray. 10. EXPOSURE: Place the gel between two cellophane sheets. Gently smooth out the air bubbles of the gel sandwich. Expose the gel sandwich to film for different lengths of time and develop the film, or expose the gel to a CCD camera. Signal can be detected up to 2 hours following the substrate step. Note: Recommended exposure times start at 1 minute. If this produces insufficient signal, then increase exposure time to 5 minutes or more. If there is too much signal, expose for 5-30 seconds. 4

Stripping and Reprobing the Gel 1. STRIPPING: Carefully open the cellophane sheets and add a few ml of ultrapure water to loosen the gel from the sheets. Remove the gel from the cellophane with a gel knife and place it in 20 ml of Restore Western Blot Stripping Buffer (Product No. 21059) for 30-60 minutes at 37 C with mild agitation. Note: Make sure that the gel is floating freely in the solution. When incubating at 37 C cover the colander device with plastic wrap or a clear film. 2. WASH STEP: Pour off the Stripping Buffer solution and wash the gel 3 x 10 minutes with 50 ml PBS/0.05% Tween -20 with mild agitation. Note: Make sure the gel is floating freely in the solution. This can be a stopping point by placing the gel in ultrapure water following the WASH STEP. 3. SUBSTRATE STEP: To ensure the gel has been completely stripped, transfer the gel into the clean incubation tray and incubate the gel in 10 ml of the UnBlot Substrate Working Reagent for 5 minutes at room temperature with mild agitation. Rinse the gel for 15 seconds with 50 ml ultrapure water. 4. EXPOSURE: Place the gel between two cellophane sheets. Gently smooth out the air bubbles of the gel sandwich. Expose the gel sandwich for 15 minutes and develop the film, or expose the gel to a CCD camera. Note: If bands are still visible, repeat Steps 1-3 above. Strip the gel for an additional 15 minutes at 37 C. If no bands are present following exposure, proceed to Step 5. 5. WASH STEP: Carefully open the cellophane sheets and add a few ml of ultrapure water to loosen the gel from the sheets. Remove the gel from the cellophane with a gel knife and wash the gel 3 x 5 minutes with 50 ml PBS/0.05% Tween -20 with mild agitation. Note: Make sure the gel is floating freely in the solution. 6. RE-PROBING: To re-probe the gel, repeat PRIMARY ANTIBODY STEP and continue through the EXPOSURE step. Gel Staining with GelCode Blue Stain Reagent (Product No. 24590) after In-Gel Immunodetection Procedures 1. Remove the gel from the cellophane and rinse 3 x 5 minutes with 50 ml ultrapure water. 2. Incubate the gel in 50 ml GelCode Blue Stain Reagent for 1 hour at room temperature. 3. Rinse the gel 2 x 5 minutes with 50 ml ultrapure water. 4. Destain the gel with 5% acetic acid/40% methanol. Change the destain solution until the gel and the solution are clear. 5. Place the gel in water until ready for preservation by the method of your choice. References 1. Burridge, K. (1976). Proc. Natl. Acad. Sci. USA 73, 4457-4461. 2. Rosta, J.A, Kelly, P.T and Cotman, C.W. (1977). Anal. Biochem. 80, 366-372. 3. Olden, K. and Yamada, K.M. (1977). Anal. Biochem. 78, 483-490. 4. Adair, W.S., Jurivich, D. and Goodenough, U.S. (1978). J. Cell Biol. 78, 281-285. 5. Towbin, H., Staehlin, T. and Gordon, J. (1979). Proc. Natl. Acad. Sci. USA 76, 4350-4354. 6. Renart, J., Reiser, J. and Stark, G.R. (1979). Proc. Natl. Acad. Sci. USA 76, 3116-3120. 7. Gersten, D.M. (1996). Gel Electrophoresis:Proteins (D. Rickwood, ed.) John Wiley and Sons. 8. Lin, W. and Kasamatsu, H. (1983). Anal. Biochem. 128, 302-311. 9. DenHollander, N. and Befus, D. (1989). J. Immunol. Meth. 122, 129-135. Tween is a registered trademark of ICI Americas. Pierce Chemical Company, 4/2001. Printed in the USA. 5

Troubleshooting SYMPTOM CAUSE REMEDY High background at the top of the gel High background High background High background Did not cut enough off the top of the gel Primary antibody concentration too high (e.g., 200 ng/ml) Secondary antibody concentration too high (e.g., 1:500 dilution or 20 ng/ml) Inadequate washes or gel was sticking to the bottom of the tray Cut 0.5-1 cm from the bottom of the comb Further dilute primary antibody (e.g., 100 ng/ml) Reduce secondary antibody concentration (e.g., Dilute 1:1000 or 10 ng/ml) Increase the length (e.g., 3 x 20 min 50 ml/wash) or frequency of washes (e.g., 6 x 5 min 50 ml/wash). Make sure the gel is floating freely in the solution during the wash step Reduce the substrate incubation and/or follow with water rinse Use the cellophane Exposure Sheets provided with the kit High background UnBlot Substrate incubation too long and/or water wash was omitted High background Plastic wrap was used to encase the gel for exposure to film High background Film exposure too long Reduce the film exposure (e.g., From 1 minute to 30 seconds or 15 seconds) Low signal Secondary antibody, HRP diluted too much Try 2- to 5-fold higher secondary antibody-hrp concentration Low signal Primary antibody diluted too much Try 2- to 5-fold higher primary antibody concentration Note: Alter the primary antibody concentration only if adjusting the secondary antibody-hrp concentration had no effect or results in high background. Diffused bands Residual alcohol remaining on gel surface following Pretreatment Step Make sure the gel is not sticking to the bottom of the tray and/or is totally submerged but freely floating during water wash Diffused bands Gel was running too fast Decrease the voltage during the electrophoresis run Diffused bands Old gel Do not use gels that have expired. Old gels more significantly affect In-Gel Detection than Western blotting. Fuzzy bands Protein degradation Make fresh protein samples Nonspecific bands Incompatible Antibody Dilution Buffer Try one of the following buffers: 0.25X-1X Blocker Casein in PBS (Product No. 37528) 1 mg/ml Goat Gamma Globulin (Product No. 31869) 1X SuperBlock Blocking Buffer (Product No. 37515) No bands Protein did not migrate from the stacker, % gel too high No bands Primary antibody is not recognizing the target protein No bands Secondary antibody is not recognizing the primary antibody Use lower % gel Be sure to select primary antibody that recognizes the target protein Change the primary antibody to recognize the secondary-hrp antibody conjugate (i.e., rabbit/antirabbit-hrp) Change the secondary-hrp antibody conjugate to recognize the primary antibody (i.e., anti-rabbit- HRP/rabbit) 6