This kit is protected by the following patents:

UK/Belgium/France/Germany/Italy/Netherlands/Sweden/Switzerland: EPO116454; Australia: 575552; Canada: 1217121;Finland: 76380; Japan: 1649 482; New Zealand: 207095; SouthAfrica: 84/0909; USA: 4598044

and is sold under licence from the patent holder (BritishTechnology Group Ltd)

ECL, ECL + device, Hybond, Hyperfilm, Hypercassette andSensitize are trademarks of Amersham Pharmacia Biotech Limited

or its subsidiariesAmersham is a trademark of Nycomed Amersham plc

Pharmacia and Drop Design are trademarks of Pharmacia & Upjohn Inc

Tween is a trademark of ICI Americas IncSaranWrap is a trademark of Dow Chemical Company

Wratten is a trademark of Kodak Ltd

© Amersham Pharmacia Biotech UK Limited 1999 – All rightsreserved

All goods and services are sold subject to the terms and conditionsof sale of the company within the Amersham Pharmacia Biotechgroup which supplies them. A copy of these terms and conditionsis available on request.

http://www.apbiotech.comAmersham Pharmacia Biotech UK Limited Amersham PlaceLittle Chalfont Buckinghamshire England HP7 9NAAmersham Pharmacia Biotech AB SE-751 84 Uppsala SwedenAmersham Pharmacia Biotech Inc 800 Centennial AvenuePO Box 1327 Piscataway NJ 08855 USAAmersham Pharmacia Biotech Europe GmbH Munzinger Strasse 9 D-79111 Freiburg Germany

RPN2106PL/99/10

ECL Western blotting detection reagentsECL Western blotting analysis system

RPN 2106RPN 2108RPN 2109RPN 2209RPN 2134

STORAGEStore at 2-8ºC

STABILITYThe reagents are stable for at least 6 monthswhen stored under the recommended conditions.

Warning: For research use only.Not recommended or intended for diagnosisof disease in humans or animals. Do not useinternally or externally in humans or animals.

RPN2106PL 99/10 03/05/00 03:53 pm Page 1

32

COMPONENTS OF THE SYSTEMSRPN 2106 ECL Western blotting detection reagentsDetection reagent 1 (250ml)Detection reagent 2 (250ml)Sufficient for 4000cm2 membrane

RPN 2209 ECL Western blotting detection reagentsDetection reagent 1 (125ml)Detection reagent 2 (125ml)Sufficient for 2000cm2 membrane

RPN 2109 ECL Western blotting detection reagentsDetection reagent 1 (62.5ml)Detection reagent 2 (62.5ml)Sufficient for 1000cm2 membrane

RPN 2134 ECL Western blotting detection reagents3 x RPN 2209Sufficient for 6000cm2 membrane

These products are for the detection of membrane boundperoxidase labelled antibodies

RPN 2108 ECL Western blotting analysis systemDetection reagent 1 (62.5ml)Detection reagent 2 (62.5ml)Mouse Ig, horseradish peroxidase-linked whole antibody (fromsheep) (100µl)Rabbit Ig, horseradish peroxidase-linked whole antibody (fromdonkey) (100µl)Blocking reagent (5g)Sufficient for 10 blots 10cmx10cm

For the detection of either mouse or rabbit membrane boundprimary antibodies.

RPN2106PL 99/10 03/05/00 03:53 pm Page 2

Contents Page

Components of the systems 3

Safety warnings and precautions 5

Description 6

Critical parameters 8

Additional equipment and reagents required 9

ECL immunodetection procedure 12Flow diagram 12Detailed protocol and notes 13Summary protocol 23

Additional procedures 27Rapid immunodetection protocol 27Determination of optimum antibody concentration 30Quantification of proteins on ECL Western blots 32Use of ECL protein molecular weight markers 36

Additional information 40Troubleshooting guide 40Quality control 47Background and references 48Related products 51

54

SAFETY WARNINGS ANDPRECAUTIONSWarning: For research use only. Not recommended or intendedfor diagnosis of disease in humans or animals. Do not useinternally or externally in humans or animals.

We recommend that this product and components are handledonly by those persons who have been trained in laboratorytechniques and that it is used in accordance with the principlesof good laboratory practice. As all chemicals should beconsidered as potentially hazardous, it is advisable whenhandling chemical reagents to wear suitable protective clothing,such as laboratory overalls, safety glasses and gloves. Careshould be taken to avoid contact with skin or eyes. In case ofcontact with skin or eyes, wash immediately with water.

You are reminded that certain components in the solutions maycause bleaching on contact with skin.

RPN2106PL 99/10 03/05/00 03:53 pm Page 4

DESCRIPTIONECL™ Western blotting from Amersham Pharmacia Biotech is alight emitting non-radioactive method for detection ofimmobilised specific antigens, conjugated directly or indirectlywith horseradish peroxidase-labelled antibodies.

● High sensitivity non-radioactive detection systemDetection of less than 1pg of antigen on Hybond™ ECL, atleast 10x more sensitive than other non-radioactive orradioactive detection systems.

● High resolutionHigh contrast signal generated

● SpeedSpecific protein detection may be achieved in less than 1minute.

● Stable hard copy results on filmSignal generated can be quantitated with a densitometer.

● Detection of low abundance protein in complex cell samples● Detection of antigen with small amount of antibody or low

affinity antibody● Versatility

Detection of Western blotted proteins from one dimensional,two-dimensional and agarose/acrylamide gels.

● Optimised protocolsReprobing; sequential reprobing of membranes with a varietyof antibodies.Stripping and reprobing; the complete removal of primaryand secondary antibodies from membranes without antigendamage.Determination of optimum antibody concentration.

76

Figu

re 1

.Pri

ncip

les

of E

CL

Wes

tern

blo

ttin

g

Seco

ndar

y A

b-H

RP

Pera

cid

Prim

ary

Ab

Lig

ht

Oxi

dise

dpr

oduc

t

Hyb

ond

EC

LH

yper

film

EC

L

Prot

ein

Oxi

dise

d fo

rmof

enz

yme

+lu

min

ol+

enha

ncer

RPN2106PL 99/10 03/05/00 03:53 pm Page 6

98

CRITICAL PARAMETERSThe following points are critical:● It is essential to optimise both primary and secondary

antibodies for results with high signal and low backgrounddue to the extreme sensitivity of the system.

● It is necessary to work quickly once the membranes havebeen exposed to the detection system.

● Wear powder-free gloves when handling film or detectionreagents.

● Do not use azide as a preservative for buffers to be used inimmunodetection as it is an inhibitor of horseradishperoxidase.

● Proper blocking and washing of the membranes is critical foroptimum results. It may be necessary to adjust blockingconditions for certain applications. Use as large a volume aspossible of washing buffer.

● Do not allow the membranes to dry out after addition ofprimary antibody.

ADDITIONAL EQUIPMENT ANDREAGENTS REQUIREDEquipmentElectrophoresis and blotting apparatus (for Western blots)Blotting membrane, recommend Amersham PharmaciaBiotech’s Hybond ECL (nitrocellulose)Orbital shakerForceps with rounded, non-serrated tipsX-ray film cassettes, recommend Amersham PharmaciaBiotech’s Hypercassette™TimerFilm, recommend Hyperfilm™ ECL, film developing facilityand reagents

ReagentsTris base (tris(hydroxymethyl)aminomethane)Sodium chlorideHydrochloric acid (1M and 5M)Tween™ 20Immunodetection reagents (if using RPN 2106 and RPN 2109)Distilled waterDisodium hydrogen orthophosphate anhydrous (Na2HPO4)Sodium dihydrogen orthophosphate (NaH2PO4.2H20)

Buffers and working solutionsBuffers

Phosphate buffered saline (PBS) pH7.5:11.5g disodium hydrogen orthophosphate anhydrous (80mM)2.96g sodium dihydrogen orthophosphate (20mM)5.84g sodium chloride (100mM)Dilute to 1000ml with distilled water - check pH.

RPN2106PL 99/10 03/05/00 03:53 pm Page 8

1110

Tris-buffered saline (TBS) pH7.6:2.42g Tris base (20mM)8g sodium chloride (137mM)3.8ml 1M hydrochloric acidDilute to 1000ml with distilled water - check pH

PBS Tween (PBS-T) and TBS Tween (TBS-T):Wash buffers and diluents. Dilute required volume of Tween 20 in the corresponding buffer. A 0.1% Tween 20concentration in PBS or TBS is suitable for most ECL Westernblotting work on nitrocellulose but concentrations carryingfrom 0.05% to 1% may be required to suit your specificrequirements.

Storage of buffer once prepared:All buffers should be stable for at least 3 months if prepared inadvance and stored at room temperature, although storage in arefrigerator (2-8ºC) may be necessary to avoid microbialspoilage.Sodium azide is not recommended for use as a bacteriocide.

Working solutions for ECL immunodetection

Membrane blocking agent:Amersham Pharmacia Biotech recommends blocking reagentsupplied or substitute with low fat dried milk dissolved in PBS-T or TBS-T; 5g per 100ml (5%).

HRP-second antibodyIt is recommended that the antibody dilution should beoptimised to maximise signal and minimise background. Ifusing the second antibodies supplied in RPN 2108, a goodstarting dilution is 1:1000. See page 31.For details of the recommended ECL HRP antibodies seepage 51.

Biotinylated antibody:It is recommended that the antibody dilution should beoptimised to suit different blotting situations. See page 30.The full range of biotinylated antibodies can be found in thecurrent Amersham Pharmacia Biotech catalogue.

Storage of working solutions once prepared:All working strength solutions should be stable for one hour atroom temperature. For longer periods it is recommended thatthey be kept in a refrigerator (2-8ºC). For reproducibleperformance equilibrate to room temperature before use.

RPN2106PL 99/10 03/05/00 03:53 pm Page 10

1312 ECL IMMUNODETECTION PROCEDURE

Flow diagram

Separate protein sample by electrophoresis

Transfer to membrane

Block non-specific sites

Incubate in primary antibody

Incubate in HRP-labelled conjugate

Incubate in biotinylatedsecond antibody

Incubate in pre-formedHRP-streptavidin complex

ECL detection reagents

Expose to film

ECL detection reagents

Expose to film

Detailed protocol and notesThe protocol outlined on the following pages has been developed in our laboratories to bethe optimum for both sensitivity and convenience. A further rapid immunodetection protocolis outlined on page 27 for situations where time is limiting. Users, however, may wish toadapt the protocols to suit their specific needs, and notes and a troubleshooting guide areprovided to assist with this.

Note: The ECL Western blotting system is extremely sensitive. For results with high signaland low background, it is essential to optimise the concentrations of both primary andsecondary antibodies. The high sensitivity means that much higher dilutions of antibodiesthan used with conventional systems are required. See page 30 for details of optimisationexperiment that can be performed to determine the best concentrations of primary andsecondary antibodies.

During immunodetection, sufficient solution should be used to adquately cover themembrane and the containers should be agitated gently on a mixer platform. When washing,the volume of wash buffer should be as large as possible; 4ml of buffer per cm2 of membraneis suggested. Brief rinses of the membrane before incubating in wash buffer will improvewashing efficiency. If exposure times of less than 5 seconds are routinely required it isrecommended that the antibodies used are further diluted as it is difficult to perform suchexposures.

RPN2106PL 99/10 03/05/00 03:53 pm Page 12

1514

Protocol

1) Performing electrophoresis and blotting

2) Blocking the membraneNon-specific binding sites are blocked byimmersing the membrane in 5% blockingreagent in Tris-buffered saline Tween (TBS-T) or phosphate buffered saline Tween

1.1) The transfer of proteins to Hybond ECL(nitrocellulose) is recommended for optimumresults. Blots may be used immediately or airdried and stored in a desiccator in arefrigerator (2-8ºC) for up to 3 months.1.2) The Hybond ECL should be pre-wettedin distilled water followed by equilibrationin transfer buffer for 5-10 minutes beforeblotting.1.3) Stored blots do not require pre-wettingprior to immunodetection.1.4) Hybond C extra (supportednitrocellulose) may also be used.1.5) Preliminary results with polyvinylmembranes have given good results, but thesystem has been optimised for nitrocellulose.

2.1) The combination of Tween andblocking reagent should be suitable for mostprotein blotting work. Optimum Tweenconcentrations will vary to suit specificexperiments, but a 0.1% Tween 20

Notes

(PBS-T) for one hour at room temperatureon an orbital shaker.

3) WashingWe recommend PBS-T or TBS-T forwashing buffer.Briefly rinse the membrane using twochanges of washing buffer then wash oncefor 15 minutes and twice for 5 minutes withfresh changes of the washing buffer at roomtemperature.

4) Dilution of primary antibodyDuring the washing step dilute the primary

concentration in PBS or TBS is suitable formost ECL Western blotting work onnitrocellulose membranes. Certainexperimental situations may requirealteration of the time and temperature ofthe blocking incubation.2.2) Alternative blocking procedures aregiven in the troubleshooting guide on pages45 and 46.2.3) Membranes may be left in the blockingsolution overnight in a refrigerator (2-8ºC)if more convenient.

3) As a general rule, as large a volume aspossible of washing buffer should be usedeach time.

4) Dilution of the primary antibody requiredto give optimum results will vary and

RPN2106PL 99/10 03/05/00 03:53 pm Page 14

1716

antibody. (See determination of optimumantibody concentration, p.30)

5) IncubationIncubate the membrane in diluted primaryantibody for 1 hour at room temperature.

6) WashingWash the membrane as detailed in step 3.

7) DilutionDuring the washing step dilute thebiotinylated antibody or the HRP labelledsecond antibody.If ECL biotinylated markers are to bedetected and the protocol is not a biotin-streptavidin system, then streptavidin inHRP conjugate (RPN 1231) at a 1:1500dilution should be added along with theHRP labelled second antibody.

should be determined for each antibodyused. These optimisation experiments maybe performed by dot blot analysis.

5) Incubation times and temperatures willvary and should be optimised for eachantibody. The conditions indicated arerecommended starting points.

7) The ECL Western blotting detectionreagents can be used with any HRP labelledsecond antibody or HRP labelledstreptavidin and biotinylated antibody. Dueto the sensitivity of the ECL Westernblotting detection reagents the dilution ofthe second antibody should be optimised togive the highest signal with minimumbackground (see p.31). If using the HRPlabelled antibody supplied in RPN 2108 a

Protocol

8) IncubationIncubate the membrane in the dilutedsecond antibody for 1 hour at roomtemperature.

9) WashingWash the membrane as detailed in step 3.

10) IncubationIf using a biotinylated second antibody,dilute the biotinylated HRP streptavidincomplex or streptavidin HRP conjugate andincubate for 45-60 minutes at roomtemperature.

11) WashingWash the membrane 1x15 minutes and 4x5minutes in fresh changes of wash buffer.

suitable starting dilution would be 1:1000.

8) Incubation times and temperatures mayvary for individual antibodies.

9) If HRP-labelled second antibody is used,proceed to step 11) for the washing step.

10) Incubation times may vary.

11) Thorough washing of the membranewill minimise background.

Notes

RPN2106PL 99/10 03/05/00 03:53 pm Page 16

1918 12) DetectionRead through this whole section before proceeding. It is necessary to work quickly once themembranes have been exposed to the detection solution. All steps can be carried out in a darkroom; it is only necessary to switch off the light after section 12.5. Equipment needed are anX-ray film cassette, a roll of SaranWrap™ (other 'cling-films' may be suitable), a timer andautoradiography film; Hyperfilm ECL (RPN 2103) is recommended.The use of gloves is strongly recommended from this stage onward to prevent hand contact onfilm, or detection reagents. If possible wear powder-free gloves as the powder can inhibit theECL detection reagents leading to blank patches on the film

Protocol

12.1) Mix an equal volume of detectionsolution 1 with detection solution 2 to givesufficient to cover the membranes.

12.2) Drain the excess buffer from the washedmembranes and place them on a piece ofSaranWrap, protein side up. Add the detectionreagent to the protein side of the membrane,so that the reagents are held by surfacetension on the surface of the membrane. Donot allow the surface of the membranes tobecome uncovered.

12.1) The final volume required is0.125ml/cm2 membrane.

Notes

12.3) Incubate for precisely 1 minute atroom temperature without agitation.

12.4) Drain off excess detection reagent andwrap membranes in SaranWrap. Gentlysmooth out air pockets.

12.5) Place the blots, protein side up, in thefilm cassette. Work as quickly as possible;minimise the delay between incubating themembranes in the detection reagent andexposing them to the film (next step).

12.6) Switch off the lights and carefullyplace a sheet of autoradiography film such as(Hyperfilm ECL) on top of the membranes,close the cassette and expose for 15 seconds.

12.7) Remove film, immediately replacewith a fresh piece of unexposed film, and

12.4) Drain off excess detection reagent byholding the membrane vertically andtouching the edge of the membrane againsttissue paper. Gently place the membrane,protein side down, on to SaranWrap. CloseSaranWrap to form an envelope avoidingpressure on the membrane.

12.5) Ensure that there is no free detectionreagent in the film cassette; the film mustnot get wet.

12.6) Do this in a dark room, using redsafelights. Do not move the film whilst it isbeing exposed.

12.7) Develop first piece of filmimmediately, and on the basis of its

RPN2106PL 99/10 03/05/00 03:53 pm Page 18

2120

reclose film cassette. appearance estimate how long to continuethe exposure of the second piece of film.Second exposures can vary from 1 minute to1 hour; this will depend on the amount oftarget protein on the membrane. Ifbackground is high the membrane may berewashed twice for 10 minutes with washbuffer and re-detected following steps 12.1-12.7 with slight loss of sensitivity. If over-exposure occurs because of high lightemission resulting from high target antigenconcentration, leave blots in the cassette for5-10 minutes before re-exposing to film.

The ECL detected blots may also beexposed to polaroid film using the ECLmini-camera (RPN 2069), which wasspecifically designed for blots generatedfrom mini-gel apparatus. The ECL mini-camera is suitable for blots up to52x77mm.

13) Reprobing membranesFollowing ECL detection it is possible to reprobe the membrane several times to either clarifyor confirm results or when small or valuable samples are being analysed(5). Sequentialreprobing of membranes with a variety of antibodies is possible following the steps below. Themembranes may be stored wet wrapped in SaranWrap in a refrigerator (2-8ºC) after eachimmunodetection.

Protocol

13.1) Wash the membrane for 2x10 minutesin TBS-T or PBS-T at room temperatureusing large volumes of wash buffer.

13.2) Block the membrane by immersing in5% blocking reagent in TBS-T or PBS-T for1 hour at room temperature.

13.3) Perform immunodetection asdescribed on pages 18-20.

13.2)Refer to note 2.2) on page 15.

Notes

Protocol Notes

RPN2106PL 99/10 03/05/00 03:53 pm Page 20

2322 14) Stripping and reprobing membranesThe complete removal of primary and secondary antibodies from membranes is possiblefollowing the method outlined below. The membranes may be stripped of bound antibodiesand reprobed several times. Membranes should be stored wet wrapped in SaranWrap in arefrigerator (2-8ºC) after each immunodetection.

Protocol

14.1) Submerge the membrane in strippingbuffer (100mM 2-mercaptoethanol, 2%sodium dodecyl sulphate, 62.5mM Tris-HClpH6.7) and incubate at 50ºC for 30 minuteswith occasional agitation.

14.2) Wash the membrane for 2x10 minutesin TBS-T or PBS-T at room temperatureusing large volumes of wash buffer.

14.3) Block the membrane by immersing in5% blocking reagent TBS-T or PBS-T for 1hour at room temperature.

14.4) Perform immunodetection asdescribed on pages 18-20.

14.1) If more stringent conditions arerequired the temperature of incubation canbe increased up to a maximum of 70°C.

14.2) Membranes may be incubated withthe ECL detection reagents and exposed tofilm to ensure removal of antibodies.

14.3) Refer to note 2.2) on page 15.

Notes

Step

1) Gel loading

2) Electrophoresis and blotting

3) Preparation for immunodetectionBlocking

Prepare samples according to individualrequirements.

Perform according to usual techniques (6-15).

Prepare a 15% solution of blocking reagentin diluent buffer.

Reagent preparation

Immunodetection

Notes1) During immunodetection sufficient dilution should be used to cover the membrane and thecontainer should be agitated gently on some form of mixer platform.2) All steps for immunodetection can be performed at room temperature.

Summary protocol

Prior to immunodetection

RPN2106PL 99/10 03/05/00 03:53 pm Page 22

2524

1) Block membrane

2) Wash

3) Primary antibodyincubation

4) Washing

Incubate membrane in solution of blocking reagent

Rinse membranetwice quickly infresh wash buffer(see page 10).Wash three timeswith fresh changesof wash buffer

Incubate membranein diluted primaryantibody solution

Rinse and washmembrane in freshchanges of washbuffer

1 hour

2 quick rinses1x15 minutes2x5 minutes

1 hour

2 quick rinses1x15 minutes2x5 minutes

For step 3):Dilute primaryantibody indiluent buffer

For step 5):Dilute biotinylatedantibody or HRPlabelled secondantibody in diluentbuffer

Step Incubation time Reagent preparation

5) Biotinylatedantibody or HRP-labelled secondantibody incubation

6) Washing

7) Streptavidin-HRPincubation

8) Washing

Incubate membranein dilutedbiotinylated or HRP-labelled secondantibody solution

For HRP-labelledsecond antibody omitstep 6) and 7)

Rinse and washmembrane in freshchanges of wash

Incubate membranein diluted complex orconjugate solution

Rinse and washmembrane in freshchanges of washbuffer

45-60 minutes

2 quick rinses1x15 minutes2x5 minutes

20-60 minutes

3 quick rinses1x15 minutes2x5 minutes

For step 7):Dilute streptavidin-biotinylated HRPcomplex or strepta-vidin HRP conjugateas required

RPN2106PL 99/10 03/05/00 03:53 pm Page 24

2726

9) Detection

10) Draining

11) Exposure

12) Film developing

13) Interpretation ofresults

14) Reprobing ifrequired (see page21)

Incubate membranein equal volumes ofdetection reagent 1and detection reagent 2

Drain off detectionreagent and wrapblots in SaranWrap

Put Hyperfilm ECLon top of blot andexpose film asrequired

1 minute

15 seconds-60 minutes

Protocol

1) Electrophoresis and blotting.

2) Blocking the membraneNon specific sites are blocked by immersingthe membrane in the blocking reagentsupplied in phosphate buffered saline Tween(PBS-T) or Tris buffered saline Tween (TBS-T) and incubating for 10 minutes at roomtemperature with agitation.

3) Dilution of the primary antibodyDuring the blocking stage dilute the primaryantibody.

1) See page 14.

2)This protocol has been optimised usingthe blocking reagent supplied at 10%(w/v).Dried milk (10%) may be used as asubstitute. Other blocking agents will needto be tested for their capacity to blockeffectively in a 10 minute incubation. Theshort block is suitable for bothnitrocellulose and high quality PVDFmembranes.

3) The optimum dilution of the primaryantibody will vary and should bedetermined for each antibody used (seepage 30).

Notes

ADDITIONAL PROCEDURES

Rapid immunodetection protocolIf time is short the following protocol allows the immunodetection using HRP-labelledantibodies to be completed in just over 2 hours, compared to 4 hours 15 minutes for thestandard protocol. If desired, the protocol can be further shortened by also optimising theprimary antibody for a shortened incubation.

Step Incubation time Reagent preparation

RPN2106PL 99/10 03/05/00 03:53 pm Page 26

2928

4) RinsingRinse the membrane in PBS-T or TBS-T for15 seconds, repeat twice.

5) IncubationIncubate the membrane in the dilutedprimary antibody for 1 hour at roomtemperature with agitation.

6) WashingWe recommend PBS-T or TBS-T for thewashing buffer. Briefly rinse the membraneusing three changes of wash buffer, thenwash twice for 10 minutes with freshchanges of washing buffer, at roomtemperature.

7) Dilution of the second antibodyDuring the wash steps dilute the HRP-labelled second antibody

5) A further shortening of theimmunodetection is possible by increasingthe primary antibody concentration,allowing a reduction in the incubation timewithout compromising sensitivity. However,those wishing to do so must optimise theirantibody for the new incubation time.

6) As large a volume of wash buffer aspossible should be used at each time. As aguide, there should be at least 4ml per cm2

of membrane present.

7.1) In order to maintain the samesensitivity as obtained with the standardmethod the secondary antibody should be

Protocol

8) IncubateIncubate the membrane in the dilutedprimary antibody for 15 minutes at roomtemperature with agitation.

9) WashWash the membrane as described in step 6.

10) DetectPerform the detection with ECL reagents asdescribed on page 18.

used at a higher concentration. As aguideline increasing the concentration byfour times should maintain the samesensitivity. However, the high dilution ofantibodies normally used for ECL Westernblotting, means that such increases inantibody concentration can be performedwithout requiring excessive amounts ofreagents.7.2) Alternatively, if very short exposures tofilm are normally performed it may be moreconvenient to keep the same concentrationof secondary antibody and simply increasethe exposure time.

Notes

RPN2106PL 99/10 03/05/00 03:53 pm Page 28

3130

Determination of optimum antibodyconcentrationDue to the sensitivity of ECL Western blotting, optimisation ofantibody concentrations is necessary to ensure the best results.Generally, lower concentrations of both primary and secondaryantibodies are required with ECL compared to colorimetricdetection.Outlined below are protocols for determining optimal antibodyconcentrations.

1) Primary antibodiesDot blots are a quick and effective method of determining theoptimum dilution of a primary antibody of unknownconcentration. Alternatively, a Western blot can be preparedand then cut into several strips. It should be noted that someantibodies may require alternative blocking and washing stepsto the ones suggested below.

1.1) Spot a suitable amount of protein sample on to anitrocellulose membrane such as Hybond ECL, and allow todry. Prepare one blot for each primary antibody dilution to betested.

1.2) Incubate in block solution for 1 hour at room temperaturewith agitation.

1.3) Wash the membranes briefly in two changes of washingbuffer then wash once for 15 minutes and twice for 5 minuteswith fresh changes of the washing buffer at room temperature.

1.4) Prepare several dilutions of primary antibody: eg. 1/100,1/500, 1/1000, 1/1500. Incubate 1 blot in each antibodydilution for 1 hour at room temperature.

1.5) Wash as detailed in step 1.3.

1.6) Dilute the secondary antibody and incubate themembranes for 1 hour at room temperature.

1.7) Wash as detailed in step 1.3.

1.8) Detect using ECL detection reagents as detailed on page18. The antibody dilution that gives the maximum signal withminimum background should be selected.

2) Secondary antibodiesFor a secondary antibody of unknown activity, a dot blot isalso effective.

2.1) Prepare dot blots and block the membranes as described in1.1 and 1.2.

2.2) Incubate in primary antibody for 1 hour at roomtemperature.

2.3) Wash as detailed in step 1.3.

2.4) Prepare several dilutions of secondary antibody: eg.1/1500, 1/3000, 1/5000, 1/10000, 1/50000. Incubate 1 blot ineach antibody dilution for 1 hour at room temperature.

2.5) Wash as detailed in step 1.3.

2.6) Detect using ECL detection reagents as detailed on page18. The antibody dilution that gives maximum signal withminimum background should be selected.

RPN2106PL 99/10 03/05/00 03:53 pm Page 30

3332

Quantification of proteins on ECL Westernblots

It has been demonstrated(17) that Hyperfilm ECL exhibits alinear response to the light produced from enhancedchemiluminescence. This relationship can be used for theaccurate quantification of proteins of ECL Western blots, usingdensitometry. The range over which the film response is linearcan be extended by pre-flashing the film prior to exposure,making quantification of lower levels of protein, in particular,more accurate. Outlined below are guidelines to enablequantification of unknown levels of protein.

1) The sample containing the protein to be quantified plus a setof standards (known amounts of the same antigen) are used toprepare a Western blot. It is suggested that at least 5 differentstandard dilutions are used. The dilution range should not begreater than one order of magnitude (see example below). It isimportant that the concentration of the protein to be quantifiedlies within the standard range. To ensure this, it may be worthrunning more than one dilution of the protein.

2) If desired, the film to be used can be pre-flashed. This isperformed using a modified flash unit such as Sensitize™ RPN2051 that has been calibrated (by adjusting its distance fromthe film), to raise the film optical density 0.1 to 0.2 OD unitsabove that of the standard film. The flash duration should be inthe region of 1msec.

3) The Western blot is detected using standard protocols andthen exposed to film. For quantification to be accurate, it isimportant that the light produced is in the linear range of thefilm. This can be achieved by making several exposures ofdifferent lengths of time. If the standard of lowest

concentration is only just visible on the film, then the light fromthe rest of the standards should be in the linear range of thefilm.

4) The films can then be scanned using a densitometer, and agraph of peak area against protein concentration plotted. Theconcentration of the protein being quantified can then be readoff this graph, taking into account any dilutions made.

Example

A dilution series of myosin (chicken gizzard) was preparedcontaining 600ng, 450ng, 300ng, 150ng and 60ng per 10µl ofloading buffer. Two further test samples in the range 60-600ngwere also prepared. Samples were electrophoresed and blottedon to Hybond ECL. Immunodetection was performed usinganti-myosin (RPN 1169) at a 1:20 dilution, anti-mouse Ig-HRP(NA 931) at a 1:3000 dilution and ECL detection reagents.

A series of exposures to Hyperfilm ECL were made and the filmon which the lowest concentration of myosin was justdetectable was used for densitometric analysis. The film wasscanned using an UltroScan XL laser densitometer (PharmaciaLKB Biotechnology) and a graph was plotted of peak area (ODunits) against myosin concentration. The concentrations of thetwo test samples were then estimated from the standard curve.

RPN2106PL 99/10 03/05/00 03:53 pm Page 32

Figure 2. ECL detection of myosin standard curve and myosintest samples.

From left to right: myosin standards 600ng, 450ng, 300ng,150ng, 60ng, myosin test samples 1,2. 15 second exposure toHyperfilm ECL.

Table 1. Peak area (OD units) for myosin standards and testsamples.

Myosin samples Peak area (OD units)

Standards 600ng 2.075450ng 1.620300ng 1.149150ng 0.692

60ng 0.200

Test samples 1 0.8652 0.476

3534

Figure 3. Peak area (OD units) against myosin concentration

Table 2. Comparison of calculated with actual concentrationfor the myosin test samples.

Test sample Actual Calculated concentration concentration

(ng) (ng)

1 240ng 2352 120ng 125

Peak

are

a (O

D u

nits

)

0 100 200 300 400 500 600

ng myosin

2.4

2.0

1.6

1.2

0.8

0.4

0

RPN2106PL 99/10 03/05/00 03:53 pm Page 34

3736

Protocol

1) Remove 1µl of markers and add to 9µl ofgel loading buffer (containing 5% 2-β-mercaptoethanol).

2) Heat to 100ºC for 4 minutes. Samplesmay be loaded on to the gel immediately,or stored temporarily on ice.

3) Load 10µl per well.

1) Prepare dilution freshly, do not store themarkers in loading buffer.

2) Do not subject the markers to more thanone denaturation.

3) A 10µl loading is sufficient to produceclearly visible bands after a 15 secondexposure using overnight blotting in Towbinbuffer(9) and standard ECL Western blottingimmunodetection protocols.

Notes

Use of ECL protein molecular weight markersThe ECL protein molecular weight markers (RPN 2107) are a mixture of six different proteinslabelled with biotin for use in Western blotting following electrophoresis on a polyacrylamidegel prepared by the method of Laemmli(18). Incubation of the blot with streptavidinhorseradish peroxidase followed by detection with the ECL Western blotting system will resultin a ladder of bands of approximately equal intensity.

4) Following electrophoresis and transfer tonitrocellulose membranes, membranes areprocessed by standard immunodetectionprotocols as outlined in the main protocolsection. If the protocol used is not a biotin-streptavidin system then streptavidin-HRP(RPN 1231) is added (1:1500) in the finalantibody incubation.

5) The membranes are then washed anddetected using ECL reagents as detailed onpage 18.

6) The volume of markers required to giveoptimum results will depend on the

4.1) It is strongly advised that milk shouldnot be included in the streptavidin-HRPincubation. The binding of streptavidin tobiotin is inhibited in the presence ofendogenous biotin in the milk, resulting in amuch decreased signal when detected byenhanced chemiluminescence.

4.2) If cross reactivity is observed betweenthe streptavidin-HRP and the proteinsamples on the blot, it is suggested that thelane containing the markers is removed andincubated in streptavidin-HRP separately.The strip can then be re-aligned with therest of the membrane for ECL detection.

6.1) The loading recommended, will giveclearly visible bands after a 15 second

RPN2106PL 99/10 03/05/00 03:53 pm Page 36

3938

electroblotting and immunodetectionconditions used and the length of exposureto film required. The exact loading will haveto be determined for each application.

exposure. If the bands take longer toappear, the probable cause is inefficienttransfer to membrane. This is most likely tobe a problem with large gels. Transfershould be overnight for tank blotting, andgreater than 1 hour for semi-dry blotting.There should be good contact between thegel and the membrane during transfer. Fortank blots the use of extra Scotch-brite padsand additional securing of the transfercassettes, with rubber bands, will improvetransfer.6.2) Conversley, if the bands produced aretoo intense or a longer exposure would bemore convenient, it is suggested that ahigher dilution of markers is used.

Figure 4. Profile of ECL protein molecularweight markers.1µg sample ECL protein molecular weightmarkers diluted with 9µl of loading buffer andrun on a 12% polyacrylamide gel for 1 hourat 150 volts, followed by electroblotting on toHybond ECL overnight at 30 volts. Processingof the blot was outlined in the ECL Westernblotting protocol, using Streptavidin-HRP(RPN 1231,1:1500 dilution) and ECLWestern blotting detection reagents. The lightemission was captured using Hyperfilm ECLfor a 15 second exposure.

Figure 5. ECL protein molecular weightmarkers calibration line.

Phosphorylase b (97400)

Bovine serum albumin (68000)

Ovalbumin (46000)

Carbonic anhydrase (31000)

Trypsin inhibitor (20100)

Lysozyme (14400)

❑ Trailing edge

▲ Leading edge

4.0 4.2 4.4 4.6 4.8 5.0Log molecular weight

Ele

ctro

phor

etic

mob

ility

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

Protocol Notes

RPN2106PL 99/10 03/05/00 03:53 pm Page 38

4140

Problem Possible cause Remedy

ADDITIONAL INFORMATION

Troubleshooting guide

1) No signal 1.1) No transfer ofproteins duringWestern blotting

1.1.1) Re-evaluate blotting procedure:Stain gels with dye(15) to check transferefficiency.

1.1.2) Stain membrane with protein stain tocheck transfer efficiency.

1.1.3) Optimise gel acrylamide concentration,time for transfer and current (if electroblotting)using molecular weight markers covering themolecular weight range expected to be blotted(molecular weight and Stoke’s radius bothaffect transfer), or use a positive controlimmunoglobulin.

1.1.4) Check that gel and membrane makeproper contact during blotting.

1.2) Proteindegradation on storageof blots prior todetection

1.3) No retention ofproteins on membranes

1.1.5) Check that gel and membrane arecorrectly orientated with respect to the anode(15).

1.1.6) Check that excess temperatures are notreached during electroblotting producingbubbles, or gel/membrane distortion, etc.

1.1.7) Check that antigenicity is not destroyedby treatment for electrophoresis (SDS, urea,boiling, etc) by dot blotting antigen before andafter treatment and immunodetect using ECLdetection reagents.

1.2) Use fresh blots.

1.3.1) Assess no transfer of proteins duringWestern blotting above.1.3.2) Use fresh supply of membrane to ensureproper hydration.

RPN2106PL 99/10 03/05/00 03:53 pm Page 40

4342

1.4) Detection system 1.4.1) Check the antigen binding capacity ofthe primary antibody using a dot blot system.1.4.2) Increase (and optimise) concentration,incubation times, and temperatures of primaryantibody.1.4.3) Low affinity primary antibody: usesolutions without Tween.1.4.4) Increase (and optimise) reagentconcentration and incubation times, for thespecific application.1.4.5) Check that the detection reagents arebeing stored correctly and used asrecommended in step 12.1) in the protocol.1.4.6) Check detection reagents are working:pre-mix small quantities of detection reagent 1and detection reagent 2 (0.5ml each) and in thedark add 1µl of HRP-labelled antibody. Visibleblue light should be produced.

2) Weak signal

3) Excessivediffuse signal

2.1) See 1) above2.2) Insufficient proteinloaded on gel2.3) Low level of signal

3.1) Overloading ofprotein

2.1) See 1) above2.2) Load more protein on the gel.

2.3.1) Pre-flashing the film will increase itssensitivity to the signal and linearise itsresponse. This does, however, require care asincreased backgrounds may result. Pre-flashinginvolves hypersensitising the film just beforeuse by pre-exposure to a short flash of light(approximately 1msec). Conventionalphotographic flash units are suitable whenattenuated with a diffuser and Wratten™ 6Bfilter, to give a flash of the required intensity toincrease the 540nm absorbance of thedeveloped film to 0.15 above that of theunexposed film.2.3.2) Expose film for an extended period (1-2hours).2.3.3) Poor protein transfer on to membrane.

3.1) Load less protein on gel.

Problem Possible cause Remedy

RPN2106PL 99/10 03/05/00 03:53 pm Page 42

4544

4) Uneven/spotted blot

5) Highbackgrounds

3.2) Improper gelconditions

3.3) Antibody concen-trations too high

4.1) Improper blottingtechnique4.2) Unevenly hydratedmembrane

4.3) Fingerprintsand/or keratincontamination

5.1) Antibody concen-trations too high

3.2) Optimise gel, electrophoresis and blottingconditions.Increase acrylamide concentration of gel.Check gel and buffer recipes.Check that no bubbles interfere with transferfrom gel to membrane.3.3) Reduce concentrations of antibodies (seepage 30).

4.1) See 1) above.

4.2.1) Use new/fresh membranes.4.2.2) Make sure that membrane is fullycovered and wetted during incubations.4.3) Avoid touching membrane. Use gloves andblunt forceps.

5.1) Both primary and secondary antibodieswill give rise to high background if used at toohigh a concentration. Antibody optimisation(see page 30) should be performed.

5.2) Contaminatedblotting equipment5.3) Contaminatedbuffers5.4) Inadequateblocking

5.2) Clean or replace all equipment.

5.3) Ensure all buffers are freshly prepared andfiltered.5.4.1) Check that blocking agent solution hasbeen made up correctly.5.4.2) Use a freshly prepared solution ofblocking agent.5.4.3) Increase concentration of blocking agent(to 10% at first).5.4.4) Include blocking agent in all detectionreagent working solutions.5.4.5) Increase Tween concentration (Caution:Tween may reduce the binding of antibodies,particularly of low affinity primary antibodies).5.4.6) Increase incubation time and/ortemperature of blocking incubation.5.4.7) Try alternative blocking agents:1-10% bovine serum albumin in TBS-T orPBS-T (freshly prepared).0.5-3% gelatine in TBS-T or PBS-T (freshlyprepared).

Problem Possible cause Remedy

RPN2106PL 99/10 03/05/00 03:53 pm Page 44

4746

5.5) Problems withmembranes

Incubation times and temperatures withalbumin and gelatine blockers will need to bedetermined for each case, starting from onehour at room temperature up to overnight at37ºC or 50ºC, or various combinationsthereof(6-13,15).1% polyvinylpyrrolidone (PVP) in TBS-T orPBS-T.5.5.1) Check that the membranes arecompletely immersed in all solutions especiallyduring washing, and that membranes hydratethoroughly.5.5.2) Use a fresh supply of membranes.5.5.3) Use high quality membranes: HybondECL (RPN 2020D or RPN 82D) is therecommended nitrocellulose membrane.5.5.4) Damage to the membrane can causenon-specific binding of the detection reagents.Handle blots carefully with gloved hands andblunt non-serrated forceps.

5.6) Inadequatewashing

5.7) Detection reagents

5.8) Over exposure

5.5.5) Use clean forceps to handle blots afterwashing.5.6.1) Increase washing times and volumes ofwash buffers.5.6.2) Add Tween to reagents if not includedalready.5.6.3) Increase concentration of Tweenblocking solution.5.7.1) Rewash blots twice for 10 minutes inwash buffer and repeat detection steps.5.7.2) Excess detection reagents in blots. Drainwell by absorbing the excess on tissue paperbefore placing blots in film cassettes.5.8.1) Expose the film for a minimum period(an initial 15 seconds exposure may be all thatis required). If exposure time is too short to beconvenient, reduce antibody concentrations(see page 30).5.8.2) Leave blots in the cassette for 5-10minutes before re-exposing to film.

Problem Possible cause Remedy

Quality controlEvery batch of ECL detection reagents is functionally tested in a Western blotting applicationto ensure minimal batch to batch variability.

RPN2106PL 99/10 03/05/00 03:53 pm Page 46

4948

Background and references

Principles of ECL detection

Luminescence is defined as the emission of light resulting fromthe dissipation of energy from a substance in an excited state.In chemiluminescence the excitation is effected by a chemicalreaction. The chemical reactions of cyclic diacylhydrazides suchas luminol have been widely used in chemical analysis(1,2) andextensively studied(3,4). One of the most clearly understoodsystems is the HRP/hydrogen peroxide catalysed oxidation ofluminol in alkaline conditions. Immediately followingoxidation, the luminol is in an excited state which then decaysto ground state via a light emitting pathway. Enhancedchemiluminescence(2) is achieved by performing the oxidationof luminol by the HRP in the presence of chemical enhancerssuch as phenols. This has the effect of increasing the lightoutput approximately 1000 fold and extending the time of lightemission. The light produced by this enhanced chemiluminescentreaction peaks after 5-20 minutes and decays slowly thereafterwith a half life of approximately 60 minutes. The maximumlight emission is at a wavelength of 428nm which can bedetected by a short exposure to blue-light sensitiveautoradiography film for example Hyperfilm ECL.

Figure 6.

Figure 3. Graph of light emission versus time, showing thedifference between chemiluminescence and ECL.

1) ISACSSON, U. and WATERMARK, G., Anal. Chim. Acta.,68, pp.339-362, 1974.2) WHITEHEAD, T. P. et al., Clin. Chem., 25, pp.1531-1546,1979.3) ROSEWELL, D. F. and WHITE, E. H., Methods inEnzymology., 57, Deluca, M. A. (Ed). Academic Press, NewYork, pp.409-423, 1978.4) MOTSENBOCKER, M. A., J. Biolum. Chemilum., 2, pp.9-16, 1968.

NH2 NH2

ECL ChemiluminescenceApprox 1h Time

▲

Lig

ht

emis

sio

n

▲

RPN2106PL 99/10 03/05/00 03:53 pm Page 48

5150

5) KAUFMANN, S. H. et al., Anal. Biochem., 161, pp.89-95,1987.6) GERSHONI, J. M. and PALADE, G.E., Anal. Biochem., 131,pp.1-15, 1983.7) TOWBIN, H. and GORDON, J., J. Immunol. Meth., 72,pp.313-340, 1984.8) PELUSO, R.W. and ROSENBERG, G.H., Anal. Biochem.,162, pp.389-398, 1987.9) TOWBIN, H. STAEHELIN, T. and GORDON, J., Proc.Natl. Acad. Sci. (USA), 76, pp.4350-4354, 1979.10) GERSHONI, J. M. and PALADE, G.E., Anal. Biochem., 124,pp.396-405, 1982.11) BURNETTE, W. M., Anal. Biochem., 112, pp.195-203,1981.12) BITTNER, M., KUPFERER, P. and MORRIS, C. F., Anal.Biochem., 102, pp.457-471, 1980.13) LIN, W. and KASAMATSU, H., Anal. Biochem., 128,pp.302-311, 1983.14) ANDREWS, A. T., Electrophoresis: Theory, Techniques andBiochemical and Clinical Applications, Second Edition inMonographs on Physical Biochemistry, edited by A.R. Peacockand W. R. Harrington, Oxford Science Publications, 1986.15) JOHNSTONE, A. and THORPE, R., Immunochemistry inPractice., Blackwell Science Publications, 1982.16) LIFE SCIENCE., 7, pp.12-13, Amersham International plc,1992.17) LAEMMLI, U.K., Nature., 227, pp.680-685, 1970.18) HOFFMAN, W. L. and JUMP, A. A., Anal. Biochem., 181,pp.318-320, 1989.

Related productsHorseradish peroxidase-labelled secondantibody conjugates:Mouse Ig, horseradish peroxidase-linked whole NA 931antibody (from sheep)Rabbit Ig, horseradish peroxidase-linked whole NA 934antibody (from donkey)Rat Ig, horseradish peroxidase-linked whole NA 932antibody (from sheep)Human Ig, horseradish peroxidase-linked whole NA 933antibody (from sheep)Mouse Ig, horseradish peroxidase-linked whole NXA931antibody (from sheep) general purpose screeningreagentMouse Ig, horseradish peroxidase-linked F(ab´)2 NA9310fragment (from sheep)Rabbit Ig, horseradish peroxidase-linked F(ab´)2 NA9340fragment (from donkey)Rat Ig, horseradish peroxidase-linked F(ab´)2 NA9320fragment (from sheep)Human Ig, horseradish peroxidase-linked F(ab´)2 NA9330fragment (from sheep)

ECL Plus western blotting reagentsECL blocking agent (40g) RPN2125ECL PLus western blotting detection reagent pack RPN2124membrane blocking reagent and HRP labelled anti mouse and anti rabbit antibodies optimized forECL plus detectionECL Plus western blotting detection reagents RPN2132(sufficient for 1000cm2 membrane)ECL Plus western blotting detection reagents RPN2133(sufficient for 3000cm2 membrane)

RPN2106PL 99/10 03/05/00 03:53 pm Page 50

ECL protein molecular weight markersSix biotinylated proteins, which when incubated RPN 2107with streptavidin-HRP and ECL Western blotting detection reagents provide a ladder covering molecular weights 14400-97400Sufficient reagents for 25 loadings

Streptavidin-horseradish peroxidase RPN 1231conjugate, 2mlStreptavidin biotinylated HRP complex, 2ml RPN 1051

ECL glycoprotein detection module RPN 2190Sufficient for 25 8x10cm membrane labellings or 50 solution labellings

ECL glycoprotein detection system RPN 2191Contains:ECL glycoprotein module RPN 2190ECL Western blotting detection reagents RPN 2209

ECL streptavidin-HRP and blocking reagent RPN 2195Sufficient for 2000cm2 membrane

ECL protein biotinylation module RPN 2202Sufficient for 25 8x10cm membrane labellings or 5x2.5mg solution labellings

ECL protein biotinylation system RPN 2203Contains:ECL protein biotinylation module RPN 2202ECL Western blotting detection reagents RPN 2209

ECL phosphorylation detection system RPN 2221Contains:ECL phosphorylation module RPN 2220ECL detection reagents RPN 2209

5352

ECL phosphorylation detection module RPN 2220Sufficient for 2000cm2 membrane

Hybond ECLHigh quality nitrocellulose membranes, recommended for use with ECLPack of 10 nitrocellulose membranes, 20x20cm RPN 2020DPack of 50 nitrocellulose discs, 82mm diameter RPN 82D

Hybond-PVDF Recommended for use with ECL PlusPack of 10 PVDF membranes, 20x20cm RPN 2020PRoll of PVDF embrane, 20x3m RPN 203P

Hyperfilm ECLPack of 25 films, 18x24cm RPN 2103Pack of 25 films, 30x40 cm RPN 2104Pack of 25 films, 10x12 inches RPN 1681Pack of 25 films, 5x7inches RPN 1674

ECL mini-camera RPN 2069A camera luminometer (using polaroid film, not supplied) specifically designed for ECL Western blots generated on a mini-gel apparatus. Five sample boats supplied. (For blots up to 52x77mm)

Sensitize pre-flash unit with protocols RPN 2051

HypercassetteHypercassette, 18x24 cm RPN 1642Hypercassette, 30x40 cm RPN 1644Hypercassette, 5x7 inches RPN 1648

Amersham Pharmacia Biotech also supplies a full range of ECLproducts for nucleic acid labelling and detection. For details pleasecontact your local Amersham Pharmacia Biotech office.

RPN2106PL 99/10 03/05/00 03:53 pm Page 52