Amersham ECL Prime Western Blotting Detection Reagent

GE Healthcare Life Sciences Instructions 28-9829-42 AC Amersham Amersham ECL Prime Western Blotting Detection Reagent RPN 2232

Table of Contents Description... 3 2 Important user information... 5 3 Handling... 6 4 Components and other materials required... 7 5 How to improve the Western Blotting... 9 6 Quality control... 3 7 Protocol... 4 8 Additional information... 2 9 Troubleshooting guide... 25 0Related products... 28 References... 34 2 Instructions 28-9829-42 AC

Description Amersham ECL Prime Western Blotting detection reagent from GE Healthcare provides an improved high sensitivity chemiluminescent method for the detection of immobilized specific antigens conjugated to Horseradish Peroxidase (HRP) labeled antibodies. Introduction The peroxide-catalyzed oxidation of Luminol generates weak chemiluminescence at 425 nm. With Amersham ECL Prime detection reagent, incorporation of a redox mediator, or enhancer, into the buffer will improve the enzyme turnover and increase the equilibrium concentration of Luminol radical anion. This will significantly improve the signal intensity and the signal duration (,2). Addition of a catalyst will improve the light signal further (3). The enhanced HRP catalyzed oxidation of Luminol is a complex, multi-step reaction: Instructions 28-9829-42 AC 3

Design and features Amersham ECL Prime detection reagent is designed to provide long signal duration (24 hours) and improved sensitivity (picogram levels). The high intensity light output means that antibodies can be diluted much further than when using other chemiluminescent substrates, see Determination of optimum antibody concentration, on page 22 for recommended dilutions, and makes detection optimal with a CCD camera, e.g LAS 4000 ImageQuant from GE Healthcare. The output light can also be detected using autoradiography film (Amersham Hyperfilm ECL). Membrane compatibility Amersham ECL Prime detection reagent is optimized for use with Amersham Hybond -P PVDF membrane where the performance compared to standard chemiluminescent substrates is most enhanced, but is also compatible with Amersham Hybond ECL nitrocellulose membrane. 4 Instructions 28-9829-42 AC

2 Important user information Intended use Amersham ECL Prime detection reagent is intended for the detection of immobilized specific antigens conjugated to Horseradish Peroxidase. Amersham ECL Prime detection reagent is intended for research use only, and shall not be used in any clinical procedures, or for diagnostic purposes. Safety notices This user documentation contains CAUTIONS concerning the safe use of Amersham ECL Prime detection reagent. See definitions below. Cautions CAUTION CAUTION indicates a hazardous situation which, if not avoided, could result in minor or moderate injury. It is important not to proceed until all stated conditions are met and clearly understood. Instructions 28-9829-42 AC 5

3 Handling Safety precautions CAUTION Hazardous substances. When using hazardous chemicals, take all suitable protective measures, such as wearing protective glasses and gloves resistant to the substances used. Follow local and/or national regulations for safe operation. It is recommended to read the Materials Safety Data Sheet (MSDS) before using Amersham ECL Prime detection reagent. Storage On receipt, all components should be stored in a refrigerator at 2ºC to 8ºC. Amersham ECL Prime detection reagent is sensitive to prolonged exposure to light. Always store the individual reagents in the light-tight containers in which they are provided. Expiry The components are stable for at least 3 months when stored under the recommended conditions. See expiry date on package. 6 Instructions 28-9829-42 AC

4 Components and other materials required Kit components The following components are included in the Amersham ECL Prime detection reagent kit. Product RPN2232 Content Solution A: Luminol solution, 50 ml. Solution B: Peroxide solution, 50 ml. Sufficient for 000 cm 2 membrane Solutions Required solutions and the chemical reagents needed for these solutions are listed below. Solution Phosphate buffered saline (PBS), ph 7.5 Tris buffered saline (TBS), ph 7.6 Diluent and wash buffer PBS Tween (PBS-T) and TBS Tween (TBS-T) Preparations Prepare.5 g Di-sodium Hydrogen Orthophosphate Anhydrous (80 mm), 2.96 g Sodium Dihydrogen Orthophosphate (20 mm), 5.84 g Sodium Chloride (00 mm). 2 Dilute to 000 ml with distilled water. 3 Check ph. Prepare 8 g Sodium Chloride, 20 ml M Tris HCl, ph 7.6. 2 Dilute to 000 ml with distilled water. 3 Check ph. Dilute the required amount of Tween 20 in the corresponding buffer. 0.% Tween 20 concentration is suitable for most blotting applications. Instructions 28-9829-42 AC 7

Storage of buffers All buffers should be stable for at least 3 months if prepared in advance and stored at room temperature, although storage in a refrigerator may be necessary to avoid microbial spoilage. Do not use sodium azide as a bacteriocide. Completed Western blot membrane Use a suitable protocol to separate proteins by electrophoresis and transfer them to a PVDF or nitrocellulose membrane. Blocking reagents Blocking reagents are typically diluted to 2% to 5% (v/v) in PBS or TBS buffer. The following blocking reagents are recommended: Amersham ECL Prime Blocking Agent (RPN48) Amersham ECL Blocking Agent (RPN225) Non-fat dry milk Bovine Serum Albumine (BSA) Immunodetection reagents Primary antibody specific to the target protein(s) HRP conjugated secondary antibody that specifically binds to the primary antibody. See Amersham ECL HRP-linked Secondary Antibodies, on page 3. Dilute the antibodies according to the recommendations in Dilution ranges, on page 22. 8 Instructions 28-9829-42 AC

5 How to improve the Western Blotting Introduction To achieve an optimal western blotting result with high signal to noise ratio and best possible sensitivity and linearity, it is important to optimize the method and to select compatible products. Consider the following to get the most out of your experiment: Sample quality and loading amount It is important that the sample is of good quality and that the amount of target protein is in detectable levels. Membrane and blocking Select membrane and blocking agent compatible with sample and antibodies. Primary and secondary antibodies Always select specific antibodies of high quality and invest some time to optimize the antibody dilution. Detection and imaging Select detection reagent according to your application need. A CCD imager offers high sensitivity and broad dynamic range and provide better quantification than X-ray film. This chapter describes products recommended for use with Amersham ECL Prime detection reagent and all other Amersham ECL Western Blotting Systems from GE Healthcare. For more information regarding these products, refer to www.gelifesciences.com/ecl. Instructions 28-9829-42 AC 9

Amersham Rainbow markers Amersham Rainbow Markers are good for confirming transfer and orientation (as the colored bands transfer to the membrane) as well as determination of molecular weight. Amersham Rainbow Markers, available as full-range (M r 2 000 to 225 000, ten proteins), high-range (M r 2 000 to 225 000, eight proteins), and low-range (M r 3 500 to 38 000). Rainbow Markers are suitable for Amersham ECL and Amersham ECL Prime. Amersham ECL DualVue Markers, optimized for use with Amersham ECL and Amersham ECL Prime, are the optimal positive controls for Western Blotting. The colored bands, which are visible both on the gel and on the membrane after transfer, serve to indicate the progress of the electrophoresis, confirm protein transfer to the membrane, and help the user orientate the blot. In addition, the bands are also detected with the chemiluminescent substrates, appearing on the film or digital image and thus serve as a control for the detection system. Amersham membranes Amersham Hybond-P is a PVDF membrane suitable for all chemiluminescent Western blotting kits. It is a robust and durable membrane suitable for stripping and re-probing and is optimal for use with Amersham ECL Prime detection reagent. Amersham Hybond-ECL is a nitrocellulose membrane compatible with all chemiluminescent Western blotting substrates. 0 Instructions 28-9829-42 AC

Transfer Wet transfer (Transfer Unit TE 62), the most commonly used transfer method, is especially efficient for large proteins. Semi-dry transfer (Transfer Units TE 70 and TE 77), is faster than wet transfer and consumes less buffer. Semi-dry transfer works well for most proteins but transfer may be less efficient for large proteins. It also reduces sensitivity for very low abundance proteins. Blocking agents Unwanted binding and the resulting background caused by the primary antibody can be minimized by choosing the best blocking agent. Compatibility of the blocking agent with the detection system is important as it will affect background. The following blocking agents are recommended: Amersham ECL Prime Blocking Agent Amersham ECL Membrane Blocking Agent BSA Blocking Agent Non-fat dry milk Instructions 28-9829-42 AC

Imagers and film The ImageQuant imager range, including ImageQuant LAS 4000 and ImageQuant LAS 4000 mini, are flexible systems that cover a wide range of imaging applications including quantitative imaging of Amersham ECL, Amersham ECL Prime, Amersham ECL Plex and gel documentation. These CCD based imagers provide an affordable option for protein detection with genuine benefits, including better quantitation than film without sacrificing sensitivity, publication grade data, and image and data archiving. Amersham Hyperfilm ECL is a trusted product for reliable qualitative chemiluminescent detection in Western blots. Amersham Western Blotting website The Western Blotting website gathers years of expertise of experimental design in one place, with a user forum supported by GE scientists and engineers. For technical help, tips, and best practices in quantitative protein detection, visit www.gelifesciences.com/western. 2 Instructions 28-9829-42 AC

6 Quality control The Amersham ECL Prime detection reagent is manufactured in compliance with our ISO 900 certified quality management system, and is in conformity with the acceptance criteria set up for the product. Instructions 28-9829-42 AC 3

7 Protocol Protocol overview Below is an overview of the Western Blotting detection protocol. Gel electrophoresis Transfer Blocking agent Primary antibody Secondary antibody Detection reagent Detection 4 Instructions 28-9829-42 AC

Electrophoresis and transfer protocol Step Action Notes Perform electrophoresis and transfer according to standard protocols. Proteins should be transferred to Amersham Hybond-P for optimum results. Amersham Hybond ECL membranes can also be used. Blots may be used immediately or stored in a desiccator at 2 C to 8 C for up to 3 months. Amersham Hybond-P should be pre-wetted in 00% methanol, washed in distilled water for 5 minutes and equilibrated in transfer buffer for at least 0 minutes before transfer. Amersham Hybond ECL should be prewetted in distilled water and equilibrated in transfer buffer for at least 0 minutes before transfer. Blocking the membrane protocol Step Action Notes 2 3 Block non-specific binding sites by immersing the membrane in a suitable blocking solution, see Blocking reagents, on page 8). Incubate for hour at room temperature on an orbital shaker. Alternatively, membranes may be left in the blocking solution overnight in a refrigerator at 2 C to 8 C, if more convenient. Briefly rinse the membrane with two changes of wash buffer (see Solutions, on page 7). The combination of non-fat dried milk and Tween should be sufficient for most applications. Optimum Tween concentrations will vary to suit specific experiments, but a 0.% Tween 20 concentration is suitable for most blotting While washing, prepare the diluted primary antibody (step, Primary antibody incubation protocol, on page 6). Instructions 28-9829-42 AC 5

Primary antibody incubation protocol Step Action Notes Dilute the primary antibody in PBS-T or TBS-T. The dilution factor should be optimized for each antibody. Optimization of the antibody dilution can be performed by dot blot analysis (see Determination of optimum antibody concentration, on page 22). Due to the high signal intensity, primary and secondary antibodies might need further dilution when detecting with film, compared to CCD detection, to avoid saturated signals. 2 3 Incubate the membrane in diluted primary antibody for hour at room temperature on an orbital shaker or over night 2 C to 8 C. Briefly rinse the membrane with two changes of wash buffer. Wash the membrane by suspending it in wash buffer and agitate for 5 minutes in room temperature. Replace wash buffer at least 4 to 6 times. Incubation times and temperatures may vary and should be optimized for each antibody. The conditions indicated are recommended starting points. While washing prepare the diluted secondary antibody (step, Secondary antibody incubation protocol, on page 6.). Secondary antibody incubation protocol Step Action Notes Dilute the HRP labeled secondary antibody or biotinylated antibody in PBS-T or TBS-T. The dilution factor should be optimized for each antibody (Determination of optimum antibody concentration, on page 22). Use either an appropriate HRP labeled secondary antibody or a biotinylated secondary antibody and the HRP labeled streptavidin bridge system. 6 Instructions 28-9829-42 AC

Step 2 3 Action Incubate the membrane in the diluted secondary antibody for hour at room temperature on an orbital shaker. Briefly rinse the membrane with two changes of wash buffer. Notes Incubation times and temperatures may vary and should be optimized for each antibody. The conditions indicated are recommended starting points. 4 Wash the membrane by suspending it in enough wash buffer to cover the membrane and agitate for 5 minutes at room temperature. Replace wash buffer at least 4 to 6 times. If using an HRP labeled secondary antibody proceed directly to Detection protocol, on page 8 after this wash procedure. If using a biotinylated antibody, while washing, prepare the diluted streptavidin HRP conjugate or complex (Streptavidin bridge incubation protocol, on page 7). Streptavidin bridge incubation protocol Step If using an HRP labeled secondary antibody, proceed directly to Detection protocol, on page 8. Action Notes 2 3 Dilute the Streptavidin HRP conjugate or Streptavidinbiotinylated HRP complex in PBS-T or TBS-T. Incubate the membrane in the dilution for 45 to 60 minutes at room temperature on an orbital shaker. Briefly rinse the membrane with two changes of wash buffer. The dilution factor should be optimized (Determination of optimum antibody concentration, on page 22). Instructions 28-9829-42 AC 7

Step 4 Action Wash the membrane by suspending it in enough wash buffer to cover the membrane and agitate for 5 minutes at room temperature. Replace wash buffer at least 4 to 6 times. Notes Detection protocol Step Two detection protocols are described in this section, one for CCD camera which is optimal with Amersham ECL Prime detection reagent and another one for using autoradiography film. CCD camera protocol Action Notes 2 3 4 Allow the detection solutions to equilibrate to room temperature before opening. Mix detection solutions A and B in a ratio of : to a working solution. The final volume of detection reagent required is 0. ml/cm 2 membrane. Drain the excess wash buffer from the washed membranes and place protein side up on a sheet of plastic wrap or other suitable clean surface. Add the mixed detection reagent on to the membrane. Incubate for 5 minutes at room temperature. If the mixed reagent is not to be used immediately protect it from exposure to the light either by wrapping in foil or storing in a dark place. The reagents should cover the entire surface of the membrane, held by surface tension on to the surface of the membrane. 8 Instructions 28-9829-42 AC

Step 5 6 Action Drain off excess detection reagent by holding the membrane gently in forceps and touching the edge against a tissue. Place the blots, protein side up on a sample tray. Notes The blots can be placed on a piece of plastic wrap, protein side up, to facilitate easy movement of the film on the sample tray. 7 Place the sample tray in the CCD camera compartment. Operate the camera according to the User documentation included with the camera. Expose the membrane for 60 seconds. Exposure time can be optimized to achieve optimal results. Recommended starting exposure time is 60 seconds. Instructions 28-9829-42 AC 9

Step Autoradiography film protocol Action Notes -4 5 6 7 8 Same as step -4 in the CCD camera protocol above. Drain off excess detection reagent by holding the membrane gently in forceps and touching the edge against a tissue. Place the blots protein side down on to a fresh piece of plastic wrap, wrap up the blots and gently smooth out any air bubbles. Place the wrapped blots, protein side up, in an x-ray film cassette. Place a sheet of autoradiography film (for example, Amersham Hyperfilm ECL) on top of the membrane. Close the cassette and expose for 5 seconds. Remove the film and replace with a second sheet of unexposed film. Develop the first piece of film immediately, and on the basis of its appearance estimate how long to continue the exposure of the second piece of film. Second exposures can vary from minute to hour. Ensure there is no free detection reagent in the cassette; the film must not get wet. This stage should be carried out in a dark room using red safe lights. Do not move the film while it is being exposed. 20 Instructions 28-9829-42 AC

8 Additional information Stripping and reprobing membranes Step The complete removal of primary and secondary antibodies from the membrane is possible following the protocol outlined below. The membranes may be stripped of bound antibodies and reprobed several times. Membranes should be stored wet wrapped in plastic wrap in a refrigerator (2 to 8 C) after each immunodetection. Action Notes 2 3 4 Submerge the membrane in stripping buffer (00 mm 2-Mercaptoethanol, 2% SDS, 62.5 mm Tris-HCl ph 6.7 ) and incubate at 50 C for 30 minutes with occasional agitation. Wash the membrane for 2 x 0 minutes in PBS-T or TBS-T at room temperature using large volumes of wash buffer. Block the membrane in suitable blocking solution for hour at room temperature. Repeat the immunodetection protocol in Chapter 7, stages from Primary antibody incubation protocol to Detection protocol. If more stringent conditions are required the incubation can be performed at 70 C or incubate for a longer time. Membranes may be incubated with Amersham ECL Prime detection reagent and exposed to film to ensure removal of antibodies. Instructions 28-9829-42 AC 2

Determination of optimum antibody concentration Due to the improved sensitivity of the Amersham ECL Prime detection reagent, optimization of antibody concentrations is recommended to ensure the best results. In general, lower concentrations of both primary and secondary antibodies are required with Amersham ECL Prime detection reagent compared to standard chemiluminescent substrates, especially when using PVDF membranes. Outlined below are protocols for determining optimal antibody concentrations. Dilution ranges The following dilution ranges are recommended: Antibody Primary Secondary Primary antibodies Dilution range from mg/ml stock solution :000 - :50000 :50000 - :250000 Dot blots are a quick and effective method of determining the optimum dilution of a primary antibody of unknown concentration. Alternatively, a Western blot can be prepared and then cut into several strips. It should be noted that some antibodies may require alternative blocking and washing steps to the ones suggested below. Step Action 2 3 Spot a suitable amount of protein sample on to a PVDF or nitrocellulose membrane and allow to air dry. Prepare one blot for each primary antibody dilution to be tested. Incubate in blocking solution for hour at room temperature with agitation. Rinse the membranes briefly with two changes of wash buffer. 22 Instructions 28-9829-42 AC

Step 4 5 6 7 8 Action Prepare a number of solutions within the recommended antibody dilution range. Incubate blot in each dilution for hour at room temperature with agitation. Briefly rinse the membrane with two changes of wash buffer. Wash the membrane by suspending it in wash buffer and agitate for 5 minutes in room temperature. Replace wash buffer at least 4 to 6 times. Dilute the secondary antibody (using only one concentration) and incubate the membranes for hour at room temperature with agitation. Rinse blots in two changes of wash buffer, then wash for x 5 minutes and 3 x 5 minutes in fresh changes of wash buffer. Detect using Amersham ECL Prime detection reagent detailed in Detection protocol, on page 8. of the protocol. The antibody dilution which gives the best signal with the minimum background should be selected. Secondary antibodies Step Action 2 3 4 Spot a suitable amount of protein sample on to a PVDF or nitrocellulose membrane and allow to air dry. Prepare one blot for each primary antibody dilution to be tested. Incubate in blocking solution for hour at room temperature with agitation. Incubate in diluted primary antibody for hour at room temperature with agitation. Briefly rinse the membrane with two changes of wash buffer. Wash the membrane by suspending it in wash buffer and agitate for 5 minutes in room temperature. Replace wash buffer at least 4 to 6 times. Instructions 28-9829-42 AC 23

Step 5 6 7 Action Prepare a number of solutions within the recommended antibody dilution range. Incubate blot in each dilution for hour at room temperature with agitation. Briefly rinse the membrane with two changes of wash buffer. Wash the membrane by suspending it in wash buffer and agitate for 5 minutes in room temperature. Replace wash buffer at least 4 to 6 times. Detect using Amersham ECL Prime detection reagent detailed in Detection protocol, on page 8 of the protocol. The antibody dilution which gives the best signal with minimum background should be selected. 24 Instructions 28-9829-42 AC

9 Troubleshooting guide Problems No signal Weak signal Possible causes / remedies Incorrect species of secondary antibody has been used. PVDF membrane not pre-wetted in methanol. Check that transfer equipment is working properly and that the correct procedure has been followed. Check protein transfer by staining the gel and/or membrane. Confirm transfer efficiency by using pre-stained Rainbow marker. Some antigens may be affected by the treatments required for electrophoresis. Target protein degradation may occur if the blots are stored incorrectly. Detection reagents may have become contaminated. - To test the detection reagent activity, in a darkroom prepare to 2 ml of detection reagent working solution in a clear test tube. Add µl of undiluted HRP-conjugated antibody solution. The solution should immediately emit a visible blue light that fades during the next severals minutes. Incorrect storage of Amersham ECL Prime detection reagent may cause a loss of signal. Bad quality and/or unspecific primary antibody. Transfer efficiency may have been poor. Insufficient protein was loaded on to the gel. The concentration of primary and secondary antibodies could be too low; optimization is required. Bad quality and/or unspecific primary antibody. Exposure time may have been too short. Instructions 28-9829-42 AC 25

Problems Excessive, diffuse signal White (negative) bands on the film Uneven, spotted background Possible causes / remedies Too much protein was loaded on to the gel. Electrophoresis and transfer protocols may need optimization. The concentrations of primary and secondary antibodies could be too high; optimization is required. Negative bands generally occur when protein target is in excess and antibody concentrations are too high. The effect is caused by substrate depletion. Blotting technique requires optimization. Areas of the blot may have dried during some of the incubations. Incorrect handling can lead to contamination on the blots and/or membrane damage which may cause non-specific signal. The blocking agent is not completely dissolved in the buffer. 26 Instructions 28-9829-42 AC

Problems High backgrounds Possible causes / remedies The concentrations of primary and secondary antibodies could be too high; optimization is required. Contamination can be transferred to the blots from electrophoresis and related equipment used in blot preparation. Transfer and incubation buffers may have become contaminated and require replacing. Insufficient blocking. The blocking agent used was not freshly prepared or was too dilute or was incompatible with the application. The level of Tween used in the blocking agent was not sufficient for the application performed. The membrane was allowed to dry during some of the incubations. Poor gel quality. Unspecific and bad quality of antibodies. Insufficient washing. - The washes were not performed for a sufficient period of time or were nor performed in a high enough volume. - There was insufficient Tween in the washes. - Insufficient changes of washes were used. The film detection of the signal was allowed to over expose. The level of signal is so high that the film has become completely overloaded. Non compatible products. Instructions 28-9829-42 AC 27

0 Related products This chapter presents a subset of related products. For more information, refer to www.gelifesciences.com/ecl. Sample preparation Product Mammalian Protein Extraction Buffer Yeast Protein Extraction Buffer Kit 2-D Quant Kit Quantity (for 500 ml) 500 assays Code no. 28-942-79 28-9440-45 80-6483-56 Vivaspin sample concentrators with 30000 molecular weight cut off (MWCO) Vivaspin 500 Vivaspin 2 Vivaspin 6 Vivaspin 20 25 x 500 μl 25 x 2 ml 25 x 6 ml 2 x 20 ml 28-9322-35 28-9322-48 28-9323-7 28-9323-6 MWCO (3000, 5000, 0000, 50000 and 00000) are available for all Vivaspin volumes. Amersham markers Product Low-Range Rainbow Molecular Weight Markers High-Range Rainbow Molecular Weight Markers Full-Range Rainbow Molecular Weight Markers Amersham ECL DualVue Western Blotting Markers Amersham ECL Plex Fluorescent Rainbow Markers Amersham ECL Plex Fluorescent Rainbow Markers Amersham ECL Western Blotting Molecular Weight Markers Quantity 250 μl 250 μl 250 μl pack (25 loadings) 20 μl 500 μl pack (25 loadings) Code no. RPN755E RPN756E RPN800E RPN80 RPN850E RPN85E RPN207 28 Instructions 28-9829-42 AC

Gel electrophoresis equipment Product Electrophoresis Systems minive Vertical Electrophoresis System minive Blot Module SE 260 Mini-Vertical Unit for two slab gels SE 600 Ruby Standard Dual Cooled Vertical Unit Power Supplies EPS 30 Power Supply EPS 2A20 Power Supply Quantity Code no. 80-648-77 80-648-96 80-649-35 80-6479-57 8-30-0 28-9202-4 Transfer equipment Product Amersham ECL Semidry Blotters Quantity Code no. TE 70 Semi-Dry Transfer Unit, 4 x 6 cm 80-620-34 TE 70 PWR Semi-Dry Transfer Unit, 4 x 6 cm -003-4 TE 77 Semi-Dry Transfer Unit, 2 x 26 cm 80-62-86 TE 77 PWR Semi-Dry Transfer Unit, 2 x 26 cm -003-42 TE 62 Transfer Cooled Unit Blotting equipment 80-6209-58 Amersham ECL Multiprobe -0033-95 Amersham ECL Multiprobe XL Including Power Supply -0033-96 Instructions 28-9829-42 AC 29

Blotting membranes Product Amersham blotting membranes Hybond ECL (20 x 20 cm) Hybond-C Extra (20 x 20 cm) Hybond-P (20 x 20 cm) Hybond-LFP (20 x 20 cm) Hybond Blotting Paper (20 x 20 cm) Whatman blotting membranes Protran BA85, 0.45 μm pore size 20 x 20 cm Protran BA83, 0.2 μm pore size 20 x 20 cm Protran BA79, 0. μm pore size 20 x 20 cm Optitran BA-S 85, 0.45 μm pore size 20 x 20 cm Optitran BA-S 83, 0.2 μm pore size 20 x 20 cm Whatman blotting papers 3MM Chr 20 x 20 cm Quantity 0 sheets 0 sheets 0 sheets 0 sheets 00 sheets 25 sheets 25 sheets 25 sheets 25 sheets 25 sheets 00 sheets Code no. RPN2020D RPN2020E RPN2020F RPN2020LFP RPN60M 0409 04039 040209 04399 043939 3030-86 Amersham Blocking agents Product ECL Prime Blocking Agent ECL Blocking Agent Quantity 40 g Code no. RPN48 RPN225 30 Instructions 28-9829-42 AC

Amersham ECL HRP-linked Secondary Antibodies Product ECL Mouse IgG, HRP-Linked Whole Ab (from sheep) ECL Mouse IgG, HRP-Linked Whole Ab (from sheep) ECL Human IgG, HRP-Linked Whole Ab (from sheep) ECL Rabbit IgG, HRP-Linked Whole Ab (from donkey) ECL Rabbit IgG, HRP-Linked Whole Ab (from donkey) ECL Rat IgG, HRP-Linked Whole Antibody (from goat) ECL Mouse IgG, HRP-Linked F(ab ) 2 Fragment (from sheep) ECL Rabbit IgG, HRP-Linked F(ab ) 2 Fragment (from donkey) ECL Rat IgG, HRP-Linked F(ab ) 2 Fragment (from goat) Quantity 00 μl ml ml 00 μl ml ml ml ml ml Code no. NA93-00UL NA93-ML NA933-ML NA934-00UL NA934-ML NA935 NA930-ML NA9340-ML NA9350 Kits and Reagent Packs Product Amersham ECL Western Blotting Detection Reagents for 4000 cm 2 membrane Amersham ECL Western Blotting System Amersham ECL Western Blotting Detection Reagents for 000 cm 2 membrane Amersham ECL Western Blotting Detection Reagents for 6000 cm 2 membrane Quantity kit Code no. RPN206 RPN208 RPN209 RPN234 Instructions 28-9829-42 AC 3

Product Amersham ECL Prime Western Blotting Detection Reagents for 000 cm 2 membrane Quantity Code no. RPN2232 Amersham ECL Plex CyDye conjugated Antibodies Product Amersham ECL Plex Western Blotting Combination Pack (Cy3, Cy5, Hybond ECL) Amersham ECL Plex Western Blotting Combination Pack (Cy3, Cy5, Hybond LFP) for two slab gels Quantity Code no. RPN998 RPN999 Autoradiography Films Product Amersham Hyperfilm ECL (5 x 7 inches) Amersham Hyperfilm ECL (8 x 24 cm) Amersham Hyperfilm ECL (8 x 0 inches) Amersham Hyperfilm ECL (24 x 30 cm) Amersham Hyperfilm ECL (35 x 43 cm) Quantity 50 sheets 50 sheets 50 sheets 50 sheets 50 sheets Code no. 28-9068-35 28-9068-36 28-9068-38 28-9068-40 28-9068-4 Imaging Systems Product ImageQuant LAS 4000 ImageQuant LAS 400 ImageQuant LAS 4000 mini Quantity Code no. 28-9558-0 28-9558- 28-9558-3 32 Instructions 28-9829-42 AC

Software and Accessories Product CD and getting started: ImageQuant TL 7.0 and IQTL SecurITy 8.0 Software package (with Getting Started Guide) Licenses for ImageQuant TL only: ImageQuant TL, single user license Quantity Licenses for ImageQuant TL v7.0 and IQTL SecurITy v8.0: ImageQuant TL 7.0 and ImageQuant TL SecurITy 8.0 -user Code no. 28-9380-94 28-9236-62 28-9332-73 Instructions 28-9829-42 AC 33

References No. 2 3 Reference Kricka, L.J., Methods Enzymol. 305, 370-390, (2000). Heindl, D. and Josel, H.P. Non-radioactive Analysis of Biomolecules, 258-26, (997). Marzocci, E., Grilli. S., Della Ciana, L., Prodi, L., Roda, A. and Mirasoli, M., Anal. Biochem., 377, 89-94, (2008). 34 Instructions 28-9829-42 AC

For local office contact information, visit www.gelifesciences.com/contact GE Healthcare Bio-Sciences AB Björkgatan 30 75 84 Uppsala Sweden www.gelifesciences.com/ecl GE, imagination at work and GE monogram are trademarks of General Electric Company. Amersham, ECL, ECL DualVue, ECL Plex, Hybond, Hyperfilm, ImageQuant, Optitran, Protran, Rainbow, and Whatman are trademarks of GE Healthcare companies. This product or portions thereof is manufactured and sold under license from Cyanagen Srl and is subject of US patent number 7855287, US patent application number 200824868 and Italian application number TO200A000580, together with other equivalent granted patents and patent applications in other countries. All third party trademarks are the property of their respective owners. 2006-20 General Electric Company All rights reserved. First published Sep. 200 All goods and services are sold subject to the terms and conditions of sale of the company within GE Healthcare which supplies them. A copy of these terms and conditions is available on request. Contact your local GE Healthcare representative for the most current information. GE Healthcare Europe GmbH Munzinger Strasse 5, D-79 Freiburg, Germany GE Healthcare UK Limited Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA, UK GE Healthcare Bio-Sciences Corp. 800 Centennial Avenue, P.O. Box 327, Piscataway, NJ 08855-327, USA GE Healthcare Japan Corporation Sanken Bldg.3-25-, Hyakunincho Shinjuku-ku, Tokyo 69-0073, Japan imagination at work 28-9829-42 AC 04/20