ecl plus western blot detection -
TRANSCRIPT
i RPN2132PL Rev-B, 2002
instructions
ECL PlusWestern BlottingDetectionReagentsAn improved non-radioactivemethod for the detection ofimmobilized specific antigensconjugated to HorseradishPeroxidase labelled antibodies.
Warning
For research use only.
Not recommended or intendedfor diagnosis of disease inhumans or animals.
Do not use internally orexternally in humans oranimals.
product codes
RPN2132RPN2133
RPN2132PL Rev-B 17/1/03 2:00 pm Page 1
● 2
HandlingStorage On receipt all componentsshould be stored in arefrigerator at 2–8 ºC.
The ECL Plus reagentsare sensitive to prolongedexposure to light. Longterm storage of theindividual reagent shouldbe in the light-tightcontainers in which theyare provided.
Expiry The components of theseproducts are stable for atleast 3 months whenstored under therecommended conditions.
Packaging The ECL Plus reagents areprovided in light-tightcontainers.
ComponentsRPN2132Solution A: ECL Plussubstrate solutioncontaining tris buffer, 100 ml.
Solution B: Stock Acridansolution in Dioxane andEthanol, 2.5 ml. See safetydata sheet supplied.
Sufficient for 1000 cm2
membrane
Page finderHandling 2
Components 2
Safety warnings and precautions 3
Other materials required 3
Description 4Chemiluminescent signal 4Chemifluorescent signal 5
Critical parameters 5
Quality control 7
Protocol 81) Electrophoresis and blotting 82) Blocking the membrane 83) Primary antibody incubation 94) Secondary antibody incubation 105) Streptavidin bridge incubation 116) Detection 11
- Chemiluminescent signal 12- Chemifluorescent signal 13
Additional information 15Stripping and reprobing membranes - chemiluminescent signal 15Stripping and reprobing membranes - chemifluorescent signal 16Determination of optimum antibody concentration 18
Troubleshooting guide 19
Related products 22
References 26
Legal 28
Product information 28
RPN2132PL Rev-B 17/1/03 2:00 pm Page 2
● 3
RPN2133RPN 2132 × 3
Sufficient for 3000 cm2
membrane
Other materialsrequiredSolutions The chemical reagentsrequired for thesesolutions are availablefrom AmershamBiosciences and aredetailed in the USBTM
Ultrapure catalogue.
● Phosphate bufferedsaline (PBS) pH7.511.5 g Di-sodiumHydrogenOrthophosphateAnhydrous (80 mM)2.96 g SodiumDihydrogenOrthophosphate (20 mM) 5.84 g Sodium Chloride(100 mM)Dilute to 1000 ml withdistilled water. Check pH
● Tris buffered saline (TBS)pH7.68 g Sodium Chloride20 ml 1 M Tris HCl, PH 7.6Dilute to 1000 ml withdistilled water. Check pH
Safety warnings and precautions
Warning: For research use only. Notrecommended or intended for diagnosis ofdisease in humans or animals. Do not useinternally or externally in humans or animals.
All chemicals should be considered aspotentially hazardous. We thereforerecommend that this product is handled onlyby those persons who have been trained inlaboratory techniques and that it is used inaccordance with the principles of goodlaboratory practice. Wear suitable protectiveclothing such as laboratory overalls, safetyglasses and gloves. Care should be taken toavoid contact with skin or eyes. In the case ofcontact with skin or eyes wash immediatelywith water. (See safety data sheet for specificadvice).
Note: that the protocol requires the use ofHydrochloric acid.
Warning: Hydrochloric acid causes burns andis an irritant. Please follow themanufacturer’s safety data sheet relating tothe safe handling and use of this material.
RPN2132PL Rev-B 17/1/03 2:00 pm Page 3
● 4
● Diluent and wash bufferPBS-Tween (PBS-T)and TBS-Tween (TBS-T)Dilute the requiredamount of Tween 20 inthe correspondingbuffer. A 0.1% Tween 20concentration is suitablefor most blottingapplications.
Storage of buffers
All buffers should bestable for at least 3months if prepared inadvance and stored atroom temperature,although storage in arefrigerator may benecessary to avoidmicrobial spoilage. Do notuse Sodium Azide as abacteriocide.
Reagents ● Immunodetection
reagents (for example,primary and secondaryantibodies)
● ECL Blocking Agent(RPN2125)
Description
The ECL Plus Western blotting detectionreagents from Amersham Biosciences providean improved non-radioactive method for thedetection of immobilized specific antigensconjugated to Horseradish Peroxidase (HRP)labelled antibodies.
Chemiluminescent signalExisting chemiluminescent detection reagents,such as ECL™ Western blotting are based onthe oxidation of the cyclic Diacylhydrazide,luminol(1,2). ECL Plus utilizes a newtechnology, developed by Lumigen Inc, basedon the enzymatic generation of an acridiniumester, which produces a more intense lightemission of longer duration(3,4).
Combined HRP and peroxide catalyzedoxidation of the Lumigen PS-3 Acridansubstrate generates thousands of acridiniumester intermediates per minute. Theseintermediates react with peroxide underslight alkaline conditions to produce asustained, high intensity chemiluminescencewith maximum emission at a wavelength of 430 nm(5) (see Figure 1). The resulting lightis detected on autoradiography film (Hyperfilm ECL) or CCD camera.
ECL Plus Western blotting detection isoptimized for use with Hybond™-P PVDFmembrane where the performance over ECL
RPN2132PL Rev-B 17/1/03 2:00 pm Page 4
● 5
Western blotting is most enhanced, but isalso compatible with Hybond ECLnitrocellulose membrane.
The sensitivity increase(6) over ECL Westernblotting may be between 4 and 20 fold,depending on the immunodetection systembeing used. In addition, the duration of signalfrom ECL Plus is extended, when used incombination with PVDF membrane, allowingsuccessful exposures to be made up to 24hours after initiation of the detectionreaction. Exposures taken at this time pointwould need to be extended to 2–3 hours.
Figure 1. Chemiluminescent reaction ofLumigen PS-3 with horseradish peroxidase
Chemifluorescent signalThe chemistry of the light producing reactionwith ECL Plus enables the reagents to bescanned on an instrument such as theMolecular Dynamics™ Storm™ 860. This is
Critical parameters● Read the entire protocol
thoroughly before usingthe kit.
● ECL Plus can be usedwith both nitrocelluloseand PVDF membranesboth of which will giveimprovements insensitivity over ECL.However theimprovement observedis likely to be moresignificant with PVDFmembranes than withnitrocellulose. Inaddition the prolongedlight output is a featureof ECL Plus detectionwith PVDF membranes.Therefore in order toachieve the best resultswith ECL Plus reagentsthe use of PVDFmembranes isrecommended.
● ECL Plus is an extremelysensitive system. Forresults showing the bestsignal to noise ratio, it isessential to optimize theconcentrations of bothprimary and secondaryantibodies. Higherdilutions of antibodiesare likely to be requiredwhen using ECL Plus inplace of ECL Western
O
F
F
F
O
H
N
CH3
O
F
F
F
O
N +
CH3
Peroxide + HRP
(Acridinium ester)
F
HO F
F
CO2
+
+
O *
N
CH3
(excited product)
H
Light
Hbuffer
Peroxide
H
RPN2132PL Rev-B 17/1/03 2:00 pm Page 5
● 6
blotting, particularly inassociation with PVDFmembranes.
● During immuno-detection, sufficientsolution should be usedto adequately cover themembrane. Containersshould be agitated gentlyon a mixer platform.
● While non-fat dried milkis stronglyrecommended as themembrane blockingagent, Gelatin, Caseinand Bovine SerumAlbumin (BSA) may alsobe used as alternativeblocking reagents withthe ECL Plus system.
● When washing, thevolume of wash buffershould be as large aspossible; 4 ml of bufferper cm2 of membrane issuggested. Brief rinsesof the membrane inwash buffer beforeincubating will improvewashing efficiency.
● It is advisable to avoidthe use of containersthat are polystyrenebased to mix ECL Plusreagents as the solutionwill turn milky andproduce a precipitate.Other types of
possible due to the generation of afluorescent intermediate in the lightproducing reaction pathway with excitationof 430 nm and emission of 503 nm. ECLPlus provides excellent sensitivity with theversatility to allow use of the same Westernblot for both film exposure and instrumentscanning for quantification.
RPN2132PL Rev-B 17/1/03 2:00 pm Page 6
● 7
containers, such aspolypropylene, poly-ethylene, polymethyl-pentene and glass areall suitable for use.
● If exposure times of lessthan 5 seconds areroutinely required, it isrecommended that theantibodies used arefurther diluted as it isdifficult to perform suchshort exposures.
● Although the workingmix of the ECL Plusreagents is stable for 2to 4 hours, it isrecommended thatreagents are mixedimmediately before use.In the event that mixedreagents need to be leftbefore use, protect fromlight by wrapping thecontainer in foil or bystoring in the dark.
● A film exposure showingsimilar sensitivity levelsto that seen initially canbe achieved 24 hoursafter substrateapplication. To do thisthe exposure timeshould be increased to 2to 3 hours. However, ifthe original signal wasvery weak, detectionmay not be possible.
Quality control
Every batch of ECL Plus is functionallytested in a Western blotting application toensure minimal batch to batch variability.
RPN2132PL Rev-B 17/1/03 2:00 pm Page 7
● 8
Protocol
❶ Electrophoresis and blotting
Protocol 1.1) Perform electrophoresis andblotting according to usualtechniques. Proteins should betransferred to Hybond-P PVDF orHybond ECL for optimum results.Blots may be used immediately orstored in a desiccator at 2–8 °C forup to 3 months.
❷ Blocking the membrane
Protocol 2.1) Block non-specific bindingsites by immersing the membranein 5% non-fat dried milk, 0.1%(v/v) Tween 20 in PBS or TBS(PBS-T or TBS-T, see page 4) for 1hour at room temperature on anorbital shaker. Alternatively,membranes may be left in theblocking solution
Notes1.1) Hybond-P PVDF should bepre-wetted in 100% methanol,washed in distilled water for 5minutes and equilibrated intransfer buffer for at least 10minutes before blotting.Hybond ECL should be pre-wetted in distilled water andequilibrated in transfer buffer forat least 10 minutes beforeblotting. ECL Plus is also suitable for usewith supported nitrocellulose suchas Hybond-C Extra. Thismembrane should be prepared asfor Hybond ECL.
Notes2.1) The combination of non-fatdried milk and Tween should besufficient for most applications.Optimum Tween concentrationswill vary to suit specificexperiments, but a 0.1% Tween20 concentration is suitable formost blotting applications.
RPN2132PL Rev-B 17/1/03 2:00 pm Page 8
● 9
Protocol overnight in a refrigerator at 2–8 °C, if more convenient.
2.2) Briefly rinse the membranewith two changes of wash buffer(see page 4).
❸ Primary antibody incubation
Protocol 3.1) Dilute the primary antibodyin PBS-T or TBS-T. The dilutionfactor should be determinedempirically for each antibody.
3.2) Incubate the membrane indiluted primary antibody for 1hour at room temperature on anorbital shaker.
3.3) Briefly rinse the membranewith two changes of wash bufferand then wash the membrane in>4 ml/cm2 of wash buffer for 15minutes at room temperature.
3.4) Wash the membrane for 3 × 5 minutes with fresh changesof wash buffer at roomtemperature.
Notes
2.2) While washing prepare thediluted primary antibody (step3.1).
Notes3.1) Optimization of the antibodydilution can be performed by dotblot analysis. (see page 17).
3.2) Incubation times andtemperatures may vary andshould be optimized for eachantibody. The conditionsindicated are recommendedstarting points.
3.4) While washing prepare thediluted secondary antibody (step4.1).
RPN2132PL Rev-B 17/1/03 2:00 pm Page 9
● 10
❹ Secondary antibody incubation
Protocol 4.1) Dilute the HRP labelledsecondary antibody or biotinylatedantibody in PBS-T or TBS-T. Thedilution factor should bedetermined empirically for eachantibody (see page 17).
4.2) Incubate the membrane in thediluted secondary antibody for 1hour at room temperature on anorbital shaker.
4.3) Briefly rinse the membranewith two changes of wash bufferand then wash the membrane in >4 ml/cm2 of wash buffer for 15 minutes at room temperature.
4.4) Rinse the membrane for 3 × 5 minutes with fresh changesof wash buffer at roomtemperature.
Notes4.1) Use either an appropriateHRP labelled secondary antibodyor a biotinylated secondaryantibody and the HRP labelledstreptavidin bridge system.
4.2) Incubation times andtemperatures may vary andshould be optimized for eachantibody. The conditionsindicated are recommendedstarting points.
4.4) If using an HRP labelledsecondary antibody proceeddirectly to step 6 (detection) afterthis wash procedure.If using a biotinylated antibody,while washing, prepare thediluted streptavidin HRPconjugate or complex (step 5.1).
RPN2132PL Rev-B 17/1/03 2:00 pm Page 10
● 11
❺ Streptavidin bridge incubation
Protocol 5.1) Dilute the Streptavidin HRPconjugate or Streptavidin-biotinylated HRP complex in PBS-T or TBS-T.
5.2) Incubate the membrane in thedilution for 45–60 minutes atroom temperature on an orbitalshaker.
5.3) Briefly rinse the membranewith two changes of wash bufferand then wash the membrane in >4 ml/cm2 of wash buffer for 15minutes at room temperature.
5.4) Rinse the membrane for 3 × 5 minutes with fresh changesof wash buffer at roomtemperature.
❻ Detection
Protocol 6.1) Remove the detection reagentsfrom storage at 2–8 °C and allowto equilibrate to room temperaturebefore opening.
6.2) Mix detection solutions A andB in a ratio of 40:1 (for example, 2ml solution A + 50 µl
Notes5.1) The dilution factor should bedetermined empirically (see page17).
Notes
6.2) If the mixed reagent is not tobe used immediately protect itfrom exposure to the light either
RPN2132PL Rev-B 17/1/03 2:00 pm Page 11
● 12
Protocol solution B). The final volume ofdetection reagent required is 0.1 ml/cm2.
6.3) Drain the excess wash bufferfrom the washed membranes andplace protein side up on a sheet ofSaranWrap™ or other suitableclean surface. Pipette the mixeddetection reagent on to themembrane.
6.4) Incubate for 5 minutes atroom temperature.
Chemiluminescent detection6.5) Drain off excess detectionreagent by holding the membranegently in forceps and touching theedge against a tissue. Place theblots protein side down on to afresh piece of SaranWrap, wrap upthe blots and gently smooth outany air bubbles.
6.6) Place the wrapped blots,protein side up, in an x-ray filmcassette.
6.7) Place a sheet ofautoradiography film (forexample, Hyperfilm ECL) on top
Notesby wrapping in foil or storing in adark place.
6.3) The reagents should coverthe entire surface of themembrane, held by surfacetension on to the surface of themembrane.
6.5) Close the SaranWrap aroundthe membrane to form anenvelope or use an alternative,suitable detection pocket. Avoid applying pressure on to themembrane.
6.6) Ensure there is no freedetection reagent in the cassette;the film must not get wet.
6.7) This stage should be carriedout in a dark room using red safelights. Do not move the film while
RPN2132PL Rev-B 17/1/03 2:00 pm Page 12
● 13
Protocol of the membrane. Close thecassette and expose for 15seconds.
6.8) Remove the film and replacewith a second sheet of unexposedfilm. Develop the first piece of filmimmediately, and on the basis ofits appearance estimate how longto continue the exposure of thesecond piece of film. Secondexposures can vary from 1 minuteto 1 hour.
Chemifluorescent detection6.9) Drain off excess detectionreagent by holding the membranegently in forceps and touching theedge against a tissue.
6.10) On the Storm Imager, placethe blot protein side down on thescanning bed. Cover with a freshpiece of SaranWrap and gentlysmooth out any air bubbles.
Notesit is being exposed.
6.8) The detected blots can alsobe exposed to Polaroid™ filmusing the ECL mini-camera (RPN 2069), which is specificallydesigned for blots generated frommini-gel apparatus. The ECLmini-camera is suitable for blotsup to 52 × 77 mm.Images can also be acquired usinga CCD camera such asImageMaster™ VDS-CL (18-1130-55).
6.10) To help minimize airbubbles, a small amount of watershould be placed on the scanningbed prior to applying the blot.The blot can be wrapped inSaranWrap for scanning, howeverany creases in the SaranWrap willbe visible on the scanned image.Other types of wrap or detection
RPN2132PL Rev-B 17/1/03 2:00 pm Page 13
● 14
Protocol
6.11) Scan using the bluefluorescence / chemifluorescencemode, 100 microns, PMT between650 and 1000 v.
Notesfolder may cause loss of signal ormay themselves fluoresce.
Ensure that the blot does not dryout during or between scans. Ifthe blot dries out, higherbackground noise will occur.
6.11) To ensure the best signalintensity, it is recommended thatthe blot should be scannedstraight after substrateapplication. However, signal willstill be visible on the followingday but at a reduced level.
RPN2132PL Rev-B 17/1/03 2:00 pm Page 14
● 15
Additional information
Stripping and reprobing membranes - Chemiluminescent signalThe complete removal of primary and secondary antibodies from themembrane is possible following the protocol outlined below. Themembranes may be stripped of bound antibodies and reprobed severaltimes. Membranes should be stored wet wrapped in SaranWrap in arefrigerator (2–8 °C) after each immunodetection.
Protocol 1) Submerge the membrane instripping buffer (100 mM 2-Mercaptoethanol, 2% SDS, 62.5mM Tris-HCl pH 6.7 ) andincubate at 50 °C for 30 minuteswith occasional agitation.
2) Wash the membrane for 2 × 10 minutes in PBS-T or TBS-T at room temperature usinglarge volumes of wash buffer.
3) Block the membrane in 5%non-fat dried milk in PBS-T orTBS-T for 1 hour at roomtemperature.
4) Repeat the immunodetectionprotocol, stages 3 to 6.
Notes1) If more stringent conditions arerequired the incubation can beperformed at 70 °C or incubate fora longer time.
2) Membranes may be incubatedwith ECL Plus detection reagentsand exposed to film to ensureremoval of antibodies.
RPN2132PL Rev-B 17/1/03 2:00 pm Page 15
● 16
Stripping and reprobing membranes - chemifluorescent signalThe complete removal of primary and secondary antibodies frommembranes is possible, but removal of the fluorescent precipitate isonly possible for PVDF membranes. The treatment required is tooharsh for nitrocellulose and will either destroy or extensively damagethe membrane.Membranes may be stripped of fluorescent signal and boundantibodies, then reprobed several times but, as with all strippingprocedures, loss of antigen may occur. Membranes should be storedwet wrapped in SaranWrap in a refrigerator (2–8 °C) after eachimmunodetection.
Protocol1) Gently agitate the membrane in100% acetonitrile for 10 minutes.
2) Submerge the membrane instripping buffer (100 M 2-Mercaptoethanol, 2% SDS, 62.5mM Tris-HCl pH 6.7) andincubate at 50 °C for 30 minuteswith occasional agitation.
3) Wash the membrane for 2 × 10 minutes using large
Notes1) If the initial signal was verystrong, remove the blot from theacetonitrile and rinse briefly inwash buffer. Re-scan the blot tocheck if any signal is still present.If there is still signal, replace theblot in acetonitrile and agitate fora further 10 minutes.
2) If more stringent conditions arerequired the incubation can beperformed at 70 °C or incubatedfor a longer time.Membranes may be incubatedwith ECL Plus detection reagentsand rescanned to ensure theremoval of antibodies.
RPN2132PL Rev-B 17/1/03 2:00 pm Page 16
● 17
Protocolvolumes of wash buffer.
4) Block the membrane in 5% non-fat dried milk in PBS-T or TBS-Tfor 1 hour at room temperature.
5) Repeat the immunodetectionprotocol, stages 3 to 6.
Determination of optimum antibody concentrationDue to the improved sensitivity of the ECL Plus detection reagents,optimization of antibody concentrations is recommended to ensure thebest results. In general, lower concentrations of both primary andsecondary antibodies are required with ECL Plus compared to ECLWestern blotting, especially when using PVDF membranes.Outlined below are protocols for determining optimal antibodyconcentrations.
1) Primary antibodiesDot blots are a quick and effective method of determining the optimumdilution of a primary antibody of unknown concentration.Alternatively, a Western blot can be prepared and then cut into severalstrips. It should be noted that some antibodies may require alternativeblocking and washing steps to the ones suggested below.1.1) Spot a suitable amount of protein sample on to a nitrocelluloseor PVDF membrane and allow to air dry. Prepare one blot for eachprimary antibody dilution to be tested.1.2) Incubate in blocking solution for 1 hour at room temperaturewith agitation.1.3) Rinse the membranes briefly with two changes of wash buffer.1.4) Prepare several dilutions of primary antibody: e.g. nitrocellulose 1/1000, 1/2500, 1/5000, 1/10 000.
Notes
RPN2132PL Rev-B 17/1/03 2:00 pm Page 17
● 18
PVDF 1/5000, 1/10 000, 1/15 000, 1/20 000.Incubate 1 blot in each dilution for 1 hour at room temperature withagitation.1.5) Rinse blots in two changes of wash buffer, then wash for 1 × 15minutes and 3 × 5 minutes in fresh changes of wash buffer.1.6) Dilute the secondary antibody (using only one concentration)and incubate the membranes for 1 hour at room temperature withagitation.1.7) Wash as detailed in step 1.5.1.8) Detect using ECL Plus detection reagents as detailed in step 6 ofthe protocol. The antibody dilution which gives the best signal withthe minimum background should be selected.
2) Secondary antibodies2.1) Prepare dot blots and block the membranes as detailed in 1.1and 1.2.2.2) Incubate in diluted primary antibody for 1 hour at roomtemperature with agitation.2.3) Wash as detailed in step 1.5.2.4) Prepare several dilutions of secondary antibody: e.g. nitrocellulose 1/10 000, 1/25 000, 1/50 000, 1/100 000,PVDF 1/25 000, 1/50 000, 1/100 000, 1/200 000.Incubate 1 blot in each dilution for 1 hour at room temperature withagitation.2.5) Wash as detailed in step 1.5.2.6) Detect using ECL Plus detection reagents as detailed in step 6 ofthe protocol. The antibody dilution which gives the best signal withminimum background should be selected.
RPN2132PL Rev-B 17/1/03 2:00 pm Page 18
● 19
1.1) Check that transfer equipment is workingproperly and that the correct procedure has beenfollowed.1.2) Check protein transfer by staining the geland/or membrane.1.3) Some antigens may be affected by thetreatments required for electrophoresis.1.4) Target protein degradation may occurif the blots are stored incorrectly.
1.5) ECL Plus detection reagents may have becomecontaminated.1.6) Incorrect storage of the ECL Plus detectionreagents may cause a loss of signal.
2.1) Transfer efficiency may have been poor.
2.2) Insufficient protein was loaded on to the gel.
2.3) The concentration of primary and secondaryantibodies could be too low; optimization isrequired.
2.4) Film exposure time may have been too short.
3.1) Too much protein was loaded on to the gel.
3.2) Electrophoresis and transfer protocols mayneed optimization.
3.3) The concentrations of primary and secondaryantibodies could be too high; optimization isrequired.
❶No signal
❷Weak signal
❸Excessive, diffuse signal
Troubleshooting guideProblems Possible causes / remedies
RPN2132PL Rev-B 17/1/03 2:00 pm Page 19
● 20
❹White (negative) bands onthe film
❺Uneven, spottedbackground
❻High backgrounds
4.1) Negative bands generally occur when proteintarget is in excess and antibody concentrations aretoo high. The effect is caused by substratedepletion.
5.1) Blotting technique requires optimization.
5.2) Areas of the blot may have dried during some of the incubations.
5.3) Incorrect handling can lead to contaminationon the blots and/or membrane damage which maycause non-specific signal.
6.1) The concentrations of primary and secondaryantibodies could be too high; optimization isrequired.
6.2) Contamination can be transferred to the blots from electrophoresis and relatedequipment used in blot preparation.
6.3) Transfer and incubation buffers may havebecome contaminated and require replacing.
6.4) The blocking agent used was not freshlyprepared or was too dilute or was incompatiblewith the application.
6.5) The level of Tween used in the blocking agentwas not sufficient for the application performed.
6.6) The membrane was allowed to dry duringsome of the incubations.
6.7) The type of membrane used was notcompatible with non-radioactive systems.
6.8) The post antibody washes were not performedfor a sufficient period of time or were norperformed in a high enough volume.
Problems Possible causes / remedies
RPN2132PL Rev-B 17/1/03 2:00 pm Page 20
● 21
Problems Possible causes / remedies
6.9) There was insufficient Tween in the postantibody washes.
6.10) Insufficient changes of post antibody washeswere used.
6.11) The film detection of the signal was allowedto over expose.
6.12) The level of signal is so high that the film hasbecome completely overloaded.
RPN2132PL Rev-B 17/1/03 2:00 pm Page 21
● 22
Related products
SDS-PAGE Electrophoresis ChemicalsSee the complete range in the catalogue
Low-range Rainbow™ MW Markers, Natural45, 30, 20.1, 14.3, 6.5, 3.5 and 2.5 kDa RPN755
High-range Rainbow MW Markers, Natural220, 97, 66, 45, 30, 20.1 and 14.3 kDa RPN756
Full-range Rainbow MW Markers, Recombinant250, 160, 105, 75, 50, 35, 30, 25, 15 and 10 kDa RPN800
ECL Western Blotting MW Markers, Biotinylated97, 66, 45, 30, 20.1 and 14.3 kDa RPN2107
Hybond ECL Membrane(nitrocellulose, pore size 0.45 µm)20 × 20 cm, pack of 10 sheets RPN2020D
Hybond ECL Membrane(nitrocellulose, pore size 0.2 µm)30 cm × 3 m, 1 roll RPN3032D
Hybond-P Membrane(PVDF, pore size 0.45 µm)20 × 20 cm, pack of 10 sheets RPN2020F
Hybond-P Membrane(PVDF, pore size 0.45 µm)20 cm × 3 m, 1 roll RPN203F
Hybond-C Extra Membrane(supported nitrocellulose, pore size 0.45 µm)20 × 20 cm, pack of 10 sheets RPN2020E
RPN2132PL Rev-B 17/1/03 2:00 pm Page 22
● 23
Hybond Blotting Paper20 × 20 cm, pack of 100 sheets RPN6101M
ECL Blocking Agent, 40 g RPN2125
Mouse IgG, Horseradish Peroxidase Linked Whole Antibody (from sheep), 1 ml and 100 µl NA931
Human IgG, Horseradish Peroxidase Linked Whole Antibody (from sheep), 1 ml NA933
Rabbit IgG, Horseradish Peroxidase Linked Whole Antibody (from donkey), 1 ml and 100 µl NA934
Rat IgG, Horseradish Peroxidase Linked Whole Antibody (from goat), 1 ml NA935
Mouse IgG, Horseradish Peroxidase Linked WholeAntibody (from sheep) General Purpose ScreeningReagent, 1 ml NXA931
Mouse IgG, Horseradish Peroxidase Linked F(ab´)2
Fragment (from sheep), 1 ml NA9310
Human IgG, Horseradish Peroxidase Linked F(ab´)2
Fragment (from sheep), 1 ml NA9330
Rabbit IgG, Horseradish Peroxidase Linked F(ab´)2
Fragment (from donkey), 1 ml NA9340
Rat IgG, Horseradish Peroxidase Linked F(ab´)2
Fragment (from goat), 1 ml NA9350
Mouse IgG, Biotinylated Whole Antibody(from sheep), 2 ml RPN1001
Human IgG, Biotinylated Whole Antibody(from sheep), 2 ml RPN1003
RPN2132PL Rev-B 17/1/03 2:00 pm Page 23
● 24
Rabbit IgG, Biotinylated Whole Antibody(from donkey), 2 ml RPN1004
Rat IgG, Biotinylated Whole Antibody(from goat), 2 ml RPN1005
Immunoprecipitation Starter Pack 17-6002-35
Streptavidin-biotinylated Horseradish Peroxidase Complex RPN1051
Streptavidin Horseradish Peroxidase Conjugate RPN1231
ECL Western Blotting System RPN2108
ECL Western Blotting Detection ReagentsFor 1000 cm2 membrane RPN2109For 2000 cm2 membrane RPN2209For 4000 cm2 membrane RPN2106For 6000 cm2 membrane RPN2134
ECL Glycoprotein Detection Module25 Membrane Reactions RPN2190Order ECL Detection Reagents separately
ECL Protein Biotinylation Module RPN2202Order ECL Detection Reagents separately
ECL Protein Biotinylation SystemFor 2000 cm2 membrane RPN2203
ECL Phosphorylation ModuleSufficient for 25 blots RPN2220Order ECL Detection Reagents separately
RPN2132PL Rev-B 17/1/03 2:00 pm Page 24
● 25
Hypercassette™18 × 24 cm RPN1164230 × 40 cm RPN1164410 × 12 inches RPN116505 × 7 inches RPN11648
Hypertorch™, Red Light Darkroom Torch RPN1620
Sensitize™ Pre-flash Unit RPN2051
Hyperfilm ECL18 × 24 cm, pack of 25 films, RPN210330 × 40 cm, pack of 25 films, RPN210410 × 12 inches, pack of 25 films, RPN16815 × 7 inches, pack of 25 films, RPN1674
Hyperprocessor™ Automatic Film Processor(not available in all countries)220/240 V RPN1700110/120 V RPN1700A
ECL Mini-camera RPN2069
ImageMaster VDS-CL, CCD Camera 18-1130-55
Storm 860 and ImageQuant 860-PC
For further details see the current Amersham BiosciencesBioDirectory or contact your local office.
RPN2132PL Rev-B 17/1/03 2:00 pm Page 25
● 26
References
1) Isacsson, U. and Watermark, G., Anal. Chim. Acta. 68,339–362,(1974).
2) Whithead, T.P. et al., Clin. Chem. 25, 1531–1546, (1979).
3) Akhaven-Tafti, H. et al., Clin. Chem. 41, 1368–1369, (1995).
4) Akhaven-Tafti, H. et al., Biolum. and Chemilum. Fundamentals andApplied Aspects, 199–202, Chichester, (1994).
5) Weeks, I. et al., Clin. Chem. 29, 1474–1479, (1983).
6) Akhaven-Tafti, H. et al., Biolum. and Chemilum. Fundamentals andApplied Aspects., 313–316, Chichester, (1994).
7) Laemmli, U.K., Nature. 227, 680–685, (1970).
8) Towbin, H. and Gordon, J., J. Immun. Methods. 72, 313–340,(1984).
RPN2132PL Rev-B 17/1/03 2:00 pm Page 26
● 27
RPN2132PL Rev-B 17/1/03 2:00 pm Page 27
● 28
Lumigen PS-3 detection reagent is manufactured forAmersham Biosciences Limited by Lumigen Inc. Thiscomponent is covered by US Patent Nos. 5,491,072and 5,593,845 and is sold under license fromLumigen Inc
● 28
LegalBioDirectory, ECL, ECLPlus, Hybond,Hypercassette, Hyperfilm,Hyperprocessor,Hypertorch, ImageMaster,Molecular Dynamics,Rainbow, Sensitize andStorm are trademarks ofAmersham BiosciencesLimited
Amersham and AmershamBiosciences aretrademarks of Amershamplc
Tween is a trademark ofICI Americas Inc
SaranWrap is a trademarkof Dow Chemical Co
Polaroid is a trademark ofPolaroid (UK) Ltd
USB is a trademark ofUSB Corp
All goods and services aresold subject to the termsand condition of sale ofthe company within theAmersham BiosciencesGroup which suppliesthem. A copy of theseterms and conditions isavailable on request.
© Amersham BiosciencesUK Limited 2002 – Allrights reserved
http://www.amershambiosciences.com
Amersham Biosciences UK Limited
Amersham Place Little Chalfont Buckinghamshire UK HP7 9NA
Amersham Biosciences AB
SE-751 84 Uppsala Sweden
Amersham Biosciences Corp
800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA
Amersham Biosciences Europe GmbH
Munzinger Strasse 9 D-79111 Freiburg Germany
Product information
Product name code
ECL Plus Western Blotting Detection Reagents1000 cm2 RPN21323000 cm2 RPN2133
Related products
see pages 22–25
RPN2132PL Rev-B 17/1/03 2:00 pm Page 28