GE Healthcare

AmershamECF Western Blotting Reagent Packs

Product Booklet

Codes: RPN5781 (mouse)RPN5782 (mouse)RPN5783 (rabbit)RPN5784 (rabbit)

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Page finder1. Legal 3

2. Handling 4 2.1. Safety warnings and precautions 4 2.2. Storage 4

3. Components of the assay system 5

4. Description 6

5. Critical Parameters 7

6. Additional solutions required 8

7. ECF western blotting reagent pack procedure 11 7.1. Storage and stability 11 7.2. Protocol 11

8. Additional information 17 8.1. Determination of optimum antibody concentration 17 8.2. Quantification of proteins 18 8.3. Stripping and reprobing membranes 19

9. Troubleshooting guide 22

10. Related products 2611. References 27

1. LegalGE and GE monogram are trademarks of General Electric Company.

Amersham, CyDye, ECF, ECL, ECL Plus, FluorImager, Storm, Gene Images, Hybond and Rainbow are trademarks of GE Healthcare companies.

USB is a trademark of USB Corporation

Tween is a trademark of ICI Americas Inc

SaranWrap is a trademark of Dow Chemical Company

Whatman is a trademark of Whatman Paper Ltd

Pbxl is a trademark of Martek Biosciences Corporation

ECF Substrate is manufactured for GE Healthcare by Europa BioProducts. This component is covered by US patent number 5,424,440 and EP 424,465 B1 and is sold under license from Promega.

© 2006 General Electric Company – All rights reserved.

GE Healthcare reserves the right, subject to any regulatory and contractual approval, if required, to make changes in specification and features shown herein, or discontinue the product described at any time without notice or obligation.

Contact your GE Healthcare representative for the most current information and a copy of the terms and conditions.

http//www.gehealthcare.com/lifesciences

GE Healthcare UK Limited. Amersham Place, Little Chalfont, Buckinghamshire, HP7 9NA UK

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2. Handling

2.1. Safety warnings and precautionsWarning: For research use only. Not recommended or intended for diagnosis of disease in humans or animals. Do not use internally or externally in humans or animals.

All chemicals should be considered as potentially hazardous. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes wash immediately with water (see safety data sheet for specifi c advice).

Warning: contains sodium azide in dilute solution.

Dispose of waste by flushing with copious amounts of water to avoid the build up of explosive metallic azides in copper and lead plumbing. The total azide present in each pack is 0.1 mg.

Note that the protocols require the use of SDS, 2-mercaptoethanol, methanol, acrylamide/bis mix, ammonium persulphate and TEMED. Warning: SDS is harmful. 2-mercaptoethanol is harmful. Methanol is toxic and flammable. Acrylamide/bis mix is toxic. Ammonium persulphate is harmful and an irritant. N,N,N’N’-Tetramethyl-ethylenediamine (TEMED) is harmful and an irritant. Please follow the manufacturers’ safety data sheets relating to the safe handling and use of these materials.

2.2. StorageStore at 2-8°C.Stable for at least 3 months.

3. Components of the systemRPN 5781• Goat anti-mouse, IgG + IgM (H+L), alkaline phosphatase linked

antibody, containing sodium azide 200 μl

• Membrane blocking agent 40 g

• ECF substrate: to be dissolved in ECF dilution buffer 36 mg

• ECF dilution buffer: Warning: contains 2.4 M diethanolamine pH 10.0 in water. 60 ml

RPN 5782Consists of 2 packs of RPN 5781

RPN 5783• Goat anti-rabbit, IgG (H+L), alkaline phosphatase linked

antibody, containing sodium azide 200 μl

• Membrane blocking agent 40 g

• ECF substrate: to be dissolved in ECF detection buffer 36 mg

• ECF substrate dilution buffer: Warning: contains 2.4 M diethanolamine pH 10.0 in water. 60 ml

RPN 5784Consists of 2 packs of RPN 5783

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4. DescriptionThe ECF™ Western blotting reagent packs for use with fluorescence scanning instrumentation, for example Molecular Dynamics FluorImager™ and Storm™, allow the immunodetection of proteins separated by SDS-PAGE (1) and electroblotted on to PVDF membrane (2) using either anti-mouse or anti-rabbit alkaline phosphatase-linked immunoglobulin, followed by addition of ECF substrate. The alkaline phosphatase catalyses the conversion of ECF substrate to a highly fluorescent product which fluoresces strongly at 540–560 nm.

Flow diagram for use of the ECF Western blotting reagent packs

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››

››

››

Transfer to membrane

Block non-specific sites

Separate protein sample byelectrophoresis

Incubate in primary antibody

Incubate in anti-species AP conjugate

Incubate in ECF substrate

Scan on fluorescent scanninginstrument

5. Critical parameters• The recommended membrane for use with the ECF Western

blotting reagent pack is Hybond™ P (PVDF). Hybond ECL™, nitrocellulose, can also be used but may give lower sensitivity and becomes brittle if allowed to dry out completely after incubation in ECF substrate.

• Once the blot has been wetted, it must not be allowed to dry out during the immunodetection steps or before incubation in ECF substrate. (If the membrane does dry out briefly rinse in methanol (PVDF only) followed by PBS or TBS buffer).

• The addition of ECF substrate to the blot requires a flat, clean surface. Smoothing SaranWrap™ on to a bench is usually sufficient. Drain excess PBS wash buffer and lay the blot, protein side down, into the ECF substrate solution.

• Once the ECF substrate incubation is complete, the blot can be scanned immediately by placing it, wet, on to the sample holder of the relevant instrument.

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6. Additional solutions requiredBuffer preparationNote: All buffers should be stable for at least 3 months.

1.5M Tris-HCl, pH 8.8Dissolve 54.5 g of Tris base in 200 ml of distilled water. Adjust to pH 8.8 using concentrated HCl. Make up total volume to 300 ml with distilled water. Store at 2–8°C.

0.5M Tris-HCl, pH6.8Dissolve 12.1 g of Tris base in 150 ml of distilled water. Adjust to pH 6.8 using concentrated HCl. Make up total volume to 200 ml with distilled water. Store at 2–8°C.

10% (w/v) sodium dodecyl sulphate (SDS)Dissolve 5 g of sodium dodecyl sulphate in 50 ml of distilled water. Mix until dissolved. Store at room temperature.

10% (w/v) bromophenol blueDissolve 10 mg in 1 ml of distilled water. Store at room temperature.

SDS-PAGE loading bufferIn a 20 ml vial mix the following;

4.2 ml water

1.0 ml 0.5 M Tris-HCl, pH 6.8

800 μl glycerol

1.6 ml 10% (w/v) SDS

400 μl 2-mercaptoethanol

20 μl 10% (w/v) bromophenol blue

Store at room temperature; protect from light.

Note: This recipe is recommended because alternative tracking dyes, or even an excess of dye, will give fluorescent bands at the gel front, which may interfere with detection of the protein of interest.

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10% (w/v) Ammonium persulphate (AMPS)Dissolve 0.1 g of ammonium persulphate in 1 ml of water. USE IMMEDIATELY.

SDS-PAGE running buffer25 mm Tris, 192 mM glycine, 0.1% (w/v) SDS.

Dissolve 15 g of Tris base, 72 g glycine, 5 g SDS in 5 litres of distilled water. Mix until dissolved. Store at room temperature.

Transfer buffer25 mM Tris, 192 mM glycine.

Dissolve 15 g of Tris base, 72 g glycine in 4 litres of distilled water.

Add 1000 ml methanol and mix thoroughly. Store at 2–8°C.

Phosphate-buffered saline (PBS), pH 7.511.5 g di-sodium hydrogen orthophosphate anhydrous (80 mM)

2.96 g sodium dihydrogen orthophosphate (20 mM)

5.84 g sodium chloride (100 mM)

Dilute to 1000 ml with distilled water - check pH. Store at room temperature.

Tris-buffered saline (TBS), pH 7.620 ml 1 M Tris-HCl pH 7.6 (20 mM)

8 g sodium chloride (137 mM)

Dilute to 1000 ml with distilled water - check pH. Store at room temperature.

PBS-Tween (PBS-T) and TBS-Tween (TBS-T)Wash buffers and diluents. Dilute required volume of Tween™ 20 in the corresponding buffer; 0.1% (v/v) Tween 20 in PBS or TBS is suitable for most fluorescent Western blotting work on PVDF membrane, but concentrations ranging from 0.05% to 1% may be required to suit your specific applications. Wash buffers should be stored at room temperature.

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Gel preparationPrepare the desired % SDS polyacrylamide gels by mixing one of the following recipes (sufficient for 20 ml gel mix);

Gel percentageComponent 8% 10% 15%

Water 9.3 ml 7.9 ml 4.6 ml

30% acrylamide/bis mix 5.3 ml 6.7 ml 10.0 ml

1.5 M Tris-HCl pH 8.8 5.0 ml 5.0 ml 5.0 ml

10% (w/v) SDS 0.2 ml 0.2 ml 0.2 ml

10 (w/v) AMPS 0.2 ml 0.2 ml 0.2 ml

TEMED 0.012 ml 0.008 ml 0.008 ml

Stacking gelSufficient for 10 ml of stacking gel;

Water 5.6 ml

30% acylamide / bis 1.7 ml

0.5M Tris-HCl, pH 6.8 2.5 ml

10% (w/v) SDS 0.1 ml

10% (w/v) AMPS 0.1 ml

TEMED 0.01 ml

Note: All chemicals listed for the buffers and mixes required can be obtained from GE Healthcare through the USB™ UltraPure chemical product range.

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7. ECF Western blotting reagent pack procedure

7.1. Storage and stabilityStore at 2–8°C, protected from light. The membrane blocking agent can be stored at room temperature.

All components are stable for at least 3 months when stored under the recommended conditions.

Note: Reconstituted ECF substrate can be stored at 2–8°C for 2-4 weeks. For longer storage, it is recommended that it is divided into aliquots and frozen. Repeated freezing and thawing should be avoided.

7.2. ProtocolIt is recommended that the protocols are read thoroughly before using the system.

During immunodetection, sufficient solution should be used to adequately cover the membrane and the containers should be agitated gently on a mixer platform. When washing, the volume of wash buffer should be as large as possible; 4 ml of buffer per cm2 of membrane is suggested. Brief rinses of the membrane before incubating in wash buffer will improve washing efficiency. All steps should be carried out at room temperature.

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Protocol

1. Performing electrophoresis and blottingSeparate proteins using SDS-PAGE electrophoresis followed by electroblotting on to Hybond P (PVDF) membrane as recommended by the equipment manufacturer.

2. Blocking the membraneNon-specific binding sites are blocked by immersing the membrane in 5% (w/v) blocking agent in TBS-T or PBS-T (see page 9) for one hour on an orbital shaker at room temperature.

Notes

1.1. The transfer of proteins to PVDF membrane is strongly advised as nitrocellulose can give a lower sensitivity and if allowed to dry out (longer than 30 minutes) it will become too brittle to handle. Allowing the blot to remain wet may result in diffusion of the signal, therefore it is recommended that nitrocellulose blots should be scanned as soon as possible after detection.

1.2. The PVDF membrane should be pre-wetted in methanol for 5 seconds and then in water for 5 minutes to remove the methanol, finally equilibrating in transfer buffer for 10–15 minutes.

2.1. The combination of Tween 20 and blocking agent should be suitable for most protein work. Optimum Tween concentrations will vary to suit specific experiments, but a 0.1% Tween 20 concentration in PBS or TBS is suitable for most fluorescent work on PVDF membrane.

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Protocol

3. WashingWe recommend TBS-T or PBS-T for washing. Briefly rinse the membrane twice with fresh changes of washing buffer, then wash 3 times - once for 15 minutes and twice for 5 minutes with fresh changes of washing buffer on an orbital shaker.

4. Dilution of the primary antibodyDuring the washing step dilute the primary antibody in PBS-T or TBS-T. The required dilution of the primary antibody to give optimum results will vary and should be determined for each antibody used (see page 17).

Notes

2.1. Continued. Certain experimental situations

may require alteration of the time and temperature of the blocking incubation.

2.2. Alternatively, membranes may be left in the blocking solution overnight at 2–8°C.

3. As a general rule, as large a volume as possible of washing buffer should be used each time.

4. When quantifying protein samples linearity can, in some cases, be improved by decreasing the primary antibody concentration. Too high a concentration may lead to saturation of binding sites which will hinder the binding of the secondary antibody and should therefore be avoided.

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Protocol

5. IncubationIncubate the membrane in the diluted primary antibody for 1 hour on an orbital shaker.

6. WashingWash the membrane as detailed in step 3.

7. Dilution of alkaline phosphatase-linked anti-species antibodyDilute the anti-mouse-AP or anti-rabbit-AP 1:10 000 in PBS-T or TBS-T.

8. IncubationIncubate the membrane in diluted secondary antibody for 1 hour on an orbital shaker.

9. WashingWash the membrane as detailed in step 3.

Notes

5. For impure antibodies the addition of blocking agent to the primary antibody incubation may improve signal to noise ratio.

7. The recommended concentration of antibody can be increased further to 1:5000 to 1:2500 to obtain maximum sensitivity or to increase the signal when the primary antibody is limiting. Note: this may lead to signal saturation and hence loss of quantification properties for the higher target levels.

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Protocol

10. ECF substrate dilutionPrepare the working stock of ECF substrate by adding the 60 ml of ECF dilution buffer, supplied, to the 36 mg of ECF substrate. Mix the bottle on a roller mixer until completely dissolved.

11. ECF substrate incubationDuring the last wash stage calculate the volume of ECF substrate required to cover the membrane using 24 μl ECF substrate/cm2 membrane. Place a piece of SaranWrap on the bench and smooth out, ensuring no air bubbles or creases exist. Pipette the required volume of ECF substrate on to the SaranWrap, drain the blot of wash buffer and then lay the blot, protein side down, on to the solution ensuring no air bubbles are trapped. Leave to incubate for 5 minutes at

Notes

10. Avoid the use of stir bars or any other mechanical devices. Once reconstituted, the ECF substrate can be stored at 2–8°C for 2–4 weeks. For longer storage, we recommend that it is divided into aliquots and frozen. Repeated freezing and thawing should be avoided.

11.1. The ECF substrate must be pipetted on to a flat surface to ensure it spreads evenly across the membrane.

11.2. Wearing gloves handle the membranes carefully by the edges with blunt non-serrated forceps.

11.3. ECF substrate incubation can be extended for up to 20 minutes to increase the signal obtained but too long an incubation will result in signal diffusion. For membranes with high levels of target the protein bands may begin to become visible, in such cases remove from the ECF substrate, drain briefly and scan immediately.

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Protocol

11.3. Continued.room temperature - ensure the blot is not moved in this period.

12. Scanning the membranePlace the membrane directly on to the sample holder of the fluorescence scanning instrument and place a clean glass plate on top of it to keep it in place. Scan using a 570 nm filter. After scanning allow PVDF membrane to dry and store at 2–8°C if required for re-probing. For nitrocellulose place into PBS-T within 30 minutes of scanning and strip within 12 hours (see page 19).

Notes

12. Refer to the scanner operating manual for full instructions. From the initial scan the PMT voltage can be varied to give the best image. For accurate quantification, it is important, at the PMT used, that the signal from the sample is not at saturation. In some case further dilution of the primary and /or secondary antibody may be required to give a less intense signal.

8. Additional information

8.1. Determination of optimum antibody concentrationOptimisation of the primary antibody concentration may be necessary to ensure the best results. The following protocol is recommended as a guide for determining the optimal antibody dilution. For further information see Tech Tip 168.

Dot blots are a quick and effective method of determining the optimal dilution of a primary antibody of unknown concentration. Alternatively, a Western blot can be made 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.

1. Spot a suitable amount of protein sample on to prewetted Hybond P (PVDF) membrane (see step 1.2. in the main protocol). Keep the membrane wet until all of the samples have been applied. This is achieved by lying the membrane on top of 2–3 sheets of filter paper which have been soaked in water. After protein samples have been spotted, remove from the wet filter papers and allow the membrane to dry. Prepare one blot for each primary antibody dilution to be tested.

2. Incubate the dot blots in blocking solution for 1 hour at room temperature with agitation.

3. Rinse the membranes briefly in two changes of washing buffer then wash 3 times - once for 15 minutes and twice for 5 minutes with fresh changes of the washing buffer at room temperature with agitation.

4. Prepare several dilutions of primary antibody. Incubate 1 blot in each antibody dilution for 1 hour at room temperature with agitation.

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5. Wash as described in step 3.

6. Dilute the anti-mouse-AP or anti-rabbit-AP antibodies 1:5000-1:10 000 and incubate the dot blots for 1 hour at room temperature with agitation.

7. Wash as detailed in step 3.

8. Detect using the ECF substrate reagents as described on page 11. The antibody dilution giving the highest signal with the lowest background should be selected. However for quantification purposes, saturation of the signal should be avoided and therefore a lower dilution of antibody may be more desirable.

8.2. Quantification of proteinsQuantification of proteins of Western blots detected directly with anti-mouse-AP or anti-rabbit-AP antibodies, for example using a Molecular Dynamics FluorImager, has been shown to give a linear response within a 25 fold dilution range, this can be improved to a 50 fold dilution range with optimisation of the system. The linear relationship between fluorescent signal and amount of protein can be used for accurate protein quantitation. Outlined below are guidelines to enable quantification of unknown levels of protein.

1. The sample containing the protein to be quantified plus a set of standards (known amounts of the same antigen) are used to prepare a Western blot. It is suggested that at least 5 different dilutions are used over a 50 fold range; the signal must not be saturated. The dilution range should not be greater than one order of magnitude. It is important to ensure that the concentration of unknown lies within the standard dilutions, therefore perform a number of dilutions of the unknown protein sample for estimation.

2. Perform the immunodetection protocol as previously described, see page 11.

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3. Scan the Western blot and quantify as described in the relevant fluorescent scanning instrument manual. Use a lower PMT or alter the dilution of the primary and/or the secondary antibodies if the signal is saturating.

4. From the graph produced, read off the value of the unknown sample and taking into account the original dilution, calculate its concentration.

8.3. Stripping and reprobing membranesFollowing ECF substrate detection it is possible to reprobe the membrane several times either to clarify or confirm results or when small amounts of valuable samples are being analysed. Subsequent reprobing of membrane with a variety of antibodies is possible as long as the target proteins can be distinguished from each other by differences in their size. If not, then it will be necessary to strip the membrane before reprobing.

The complete removal of primary and secondary antibodies from dried membranes is possible following the method outlined below. If membranes are to be stripped and reprobed several times, it is suggested that the least abundant antigen is detected first to allow for target loss during the stripping process. Membranes should be stored dry, at 2–8°C, after each immunodetection; therefore it is recommended to use PVDF membrane for its robustness.

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Protocol

1. Removal of ECF reaction product

1.1. Submerge the membrane in methanol and incubate at room temperature with agitation for 30 minutes.

Notes

1.1. With nitrocellulose do not exceed 40% methanol as higher percentages of methanol may damage the membrane.

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Protocol

1.2. Rinse the membrane twice in PBS or TBS to remove all methanol.

2. Removal of antibodies2.1. Submerge the membrane

in stripping buffer (100 mM 2-mercaptoethanol, 2% (w/v) sodium dodecyl sulphate, 62.4 mM Tris-HCl, pH 6.7) (3) and incubate at 50°C for 30 minutes with agitation.

2.2. Wash the membrane for at least 2 x 15 minutes in TBS-T or PBS-T at room temperature using large volumes of wash buffer.

Notes

1.1. Continued. The methanol step is

required to remove the precipitate from the membrane.

2.1. Time and temperature are the two factors which affect the stripping. For more stringent conditions the temperature can be increased up to 70°C or the blot incubated for longer periods (up to

60 minutes). However, it must be remembered that these conditions will also remove more target protein, therefore a compromise between the two must be obtained.

2.2. It is advised to wash the blots at this stage for up to an hour, as this will help to reduce problems with background.

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Protocol

3. Reprobing3.1. Block the membrane by

immersing in 5% blocking reagent TBS-T or PBS-T for 1 hour on an orbital shaker at room temperature.

3.2. Perform immunodetection as described on page 11.

3.3. For those wishing to perform multiple stripping and reprobing simply repeat this protocol.

Notes

3.3. Multiple stripping and reprobing is best performed on dry PVDF membrane. Results have shown that the majority of target loss is during the first stripping incubation. It is suggested that the first detection cycle should be used for the least abundant antigen.

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Possible cause

1. No transfer of proteins during Western blotting

2. No retention of proteins on membranes

3. Immunodetection systems

Remedy

1. Re-evaluate blotting procedure: Stain gels with dye(4) to check transfer efficiency. Stain membrane with protein stain to check transfer efficiency. Optimise gel acrylamide concentration, time for transfer and current. Check that the gel and membrane make proper contact during blotting. Check that the gel and membrane are correctly orientated with respect to the anode (1). Check that excess temperatures are not reached during electroblotting producing bubbles, or gel/membrane distortion.

2.1. Assess transfer of proteins during Western blotting (as above).

2.2. Use a fresh supply of membrane to ensure proper hydration.

3.1. Check that the antigenicity of your protein sample is not destroyed by treatment for electrophoresis (SDS, boiling, etc.) by dot blotting antigen before and after treatment and immunodetecting using the ECF detection reagents.

3.2. Check the antigen binding capacity of the primary antibody using a dot blot system.

9. Troubleshooting guideProblem: No signal

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Possible cause

Possible cause

1. Insufficient protein loaded on gel.

2. Low level of signal.

Remedy

3.3. Increase (and optimise) concentration, incubation times and temperature of primary antibody.

3.4. Low affinity primary antibody: Use solutions without Tween 20.

3.5. Increase (and optimise) reagent concentration and incubation times, for your specific application.

3.6. Check that the ECF reagents are being stored correctly and used as recommended in the main protocol.

Remedy

1. Load more protein on gel.

2.1. Optimise the primary antibody concentration (see page 17).

2.2. Signal can be improved by increasing the concentration of the AP-linked secondary antibody from 1:10000 to 1:2500.

2.3. Scan the blot at a higher PMT voltage and/or high sensitivity settings. Refer to the instrument manual for further details.

2.4. ECF incubation can be extended for up to 20 minutes to increase the signal obtained.

Problem: Weak signal

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Possible cause

1. Overloading of protein.

2. Improper gel conditions.

3. Antibody concentrations too high.

4. ECF incubation too long.

Possible cause

1. Antibody concentrations too high.

2. Contaminated blotting equipment or buffers.

3. Inadequate blocking.

Remedy

1. Load less protein on to the gel.

2. Optimise gel electrophoresis conditions: Increase acrylamide concentration of gel. Check gel and buffer recipes. Check that no bubbles interfere with transfer from gel to membrane.

3. Reduce concentrations of antibodies.

4. Reduce the ECF incubation to prevent an excessive build up of fluorescent product, especially for high target applications.

Remedy

1. High primary antibody concentration can give rise to high backgrounds. Antibody optimisation should be performed (see page 17).

2. Clean equipment. Prepare fresh buffers for immunodetection.

3.1. Check that the blocking reagent has been made up properly.

3.2. Use freshly prepared blocking reagent.

Problem: Excessive diffuse signal

Problem: High backgrounds

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Possible cause

4. Inadequate washing

5. Problems with membranes.

Possible cause

1. Incorrect removal of excess substrate.

Remedy

3.3. Increase the concentration of blocking reagent (to 10% at first).

3.4. Try alternative blocking reagents, e.g. 1–10% BSA, 0.5–3% gelatin.

3.5. Increase incubation time and/or temperature of blocking incubation.

4.1. Increase washing times and volumes of wash buffers.

4.2. Add Tween to reagents if not already included.

4.3. Increase the concentration of Tween (note: signal may be reduced).

5.1. Ensure that PVDF membrane is sufficiently hydrated prior to use.

5.2. Ensure that membranes are completely immersed in all solutions during immunodetection.

Remedy

1. Removing excess substrate by excessive draining or placing the wet membrane directly on filter paper may sometimes result in a double band artefact. After a short drain, the membrane should be placed wet, protein side down, directly on to the scanner sample holder.

Problem: Double band artefact

10. Related productsECF Western blotting kit RPN 5780

ECF substrate for Western blotting RPN 5785

Range of fluorescein, Pbxl™ and CyDye™ see catalogueconjugated secondary antibodies for direct for detailsdetection

Full Range Rainbow™ RPN 800protein molecular weight markers

Hybond P (PVDF) Pack of 10 sheets, 20 x 20 cm RPN 2020F

Roll of membrane, 20 cm x 3 m RPN 203F

Hybond ECL (nitrocellulose)Pack of 10 sheets, 20 x 20 cm RPN 2020D

50 discs RPN 82D

For further details on these products and for information on products for ECL and ECL Plus™ detection for Western blotting, see the current GE Healthcare catalogue or contact your local GE Healthcare office.

ECF Western blotting reagent packsFor detection of mouse antibody Sufficient reagentsfor detection of 2500 cm2 of membrane RPN 5781

Sufficient reagents for detection of 5000 cm2 of membrane RPN 5782

For detection of rabbit antibody Sufficient reagentsfor detection of 2500 cm2 of membrane RPN 5783

Sufficient reagents for detection of 5000 cm2 of membrane RPN 5784

Refill pack ECF substrate for Western blotting RPN 5785

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11. References1. LAEMMLI, U.K., Nature, 227, pp.680-685, 1970.

2. TOWBIN, H. and GORDON, J., J. Immunol. Meth., 72, pp.313-340, 1984.

3. KAUFMAN, S., EWING, C. and SHAPER, J., Analyt. Biochem., 161, pp.89-95, 1987.

4. JOHNSTONE, A. and THORPE, R., Immunochemistry in Practice, Blackwell Science Publications, 1982.

imagination at work

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RPN5781PL Rev B 2006