ECL Western blotting detection reagents ECL Western blotting analysis system RPN 2106 RPN 2108 RPN 2109 RPN 2209 RPN 2134

Transcription

1 This kit is protected by the following patents: UK/Belgium/France/Germany/Italy/Netherlands/Sweden/ Switzerland: EPO116454; Australia: ; Canada: ; Finland: 76380; Japan: ; New Zealand: ; South Africa: 84/0909; USA: and is sold under licence from the patent holder (British Technology Group Ltd) ECL, ECL + device, Hybond, Hyperfilm, Hypercassette and Sensitize are trademarks of Amersham Pharmacia Biotech Limited or its subsidiaries Amersham is a trademark of Nycomed Amersham plc Pharmacia and Drop Design are trademarks of Pharmacia & Upjohn Inc Tween is a trademark of ICI Americas Inc SaranWrap is a trademark of Dow Chemical Company Wratten is a trademark of Kodak Ltd Amersham Pharmacia Biotech UK Limited 1999 All rights reserved All goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Pharmacia Biotech group which supplies them. A copy of these terms and conditions is available on request. Amersham Pharmacia Biotech UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Pharmacia Biotech AB SE Uppsala Sweden Amersham Pharmacia Biotech Inc 800 Centennial Avenue PO Box 1327 Piscataway NJ USA Amersham Pharmacia Biotech Europe GmbH Munzinger Strasse 9 D Freiburg Germany RPN2106PL/99/10 ECL Western blotting detection reagents ECL Western blotting analysis system RPN 2106 RPN 2108 RPN 2109 RPN 2209 RPN 2134 STORAGE Store at 2-8ºC STABILITY The reagents are stable for at least 6 months when stored under the recommended conditions. Warning: 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.

2 COMPONENTS OF THE SYSTEMS RPN 2106 ECL Western blotting detection reagents Detection reagent 1 (250ml) Detection reagent 2 (250ml) Sufficient for 4000cm 2 membrane RPN 2209 ECL Western blotting detection reagents Detection reagent 1 (125ml) Detection reagent 2 (125ml) Sufficient for 2000cm 2 membrane RPN 2109 ECL Western blotting detection reagents Detection reagent 1 (62.5ml) Detection reagent 2 (62.5ml) Sufficient for 1000cm 2 membrane RPN 2134 ECL Western blotting detection reagents 3 x RPN 2209 Sufficient for 6000cm 2 membrane These products are for the detection of membrane bound peroxidase labelled antibodies RPN 2108 ECL Western blotting analysis system Detection reagent 1 (62.5ml) Detection reagent 2 (62.5ml) Mouse Ig, horseradish peroxidase-linked whole antibody (from sheep) (100µl) Rabbit Ig, horseradish peroxidase-linked whole antibody (from donkey) (100µl) Blocking reagent (5g) Sufficient for 10 blots 10cmx10cm For the detection of either mouse or rabbit membrane bound primary antibodies. 3

3 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 12 Flow diagram 12 Detailed protocol and notes 13 Summary protocol 23 Additional procedures 27 Rapid immunodetection protocol 27 Determination of optimum antibody concentration 30 Quantification of proteins on ECL Western blots 32 Use of ECL protein molecular weight markers 36 Additional information 40 Troubleshooting guide 40 Quality control 47 Background and references 48 Related products 51 SAFETY WARNINGS AND PRECAUTIONS Warning: 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. We recommend that this product and components are 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. As all chemicals should be considered as potentially hazardous, it is advisable when handling chemical reagents to wear suitable protective clothing, such as laboratory overalls, safety glasses and gloves. Care should be taken to avoid contact with skin or eyes. In case of contact with skin or eyes, wash immediately with water. You are reminded that certain components in the solutions may cause bleaching on contact with skin. 4 5

4 DESCRIPTION ECL Western blotting from Amersham Pharmacia Biotech is a light emitting non-radioactive method for detection of immobilised specific antigens, conjugated directly or indirectly with horseradish peroxidase-labelled antibodies. High sensitivity non-radioactive detection system Detection of less than 1pg of antigen on Hybond ECL, at least 10x more sensitive than other non-radioactive or radioactive detection systems. High resolution High contrast signal generated Speed Specific protein detection may be achieved in less than 1 minute. Stable hard copy results on film Signal 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 protocols Reprobing; sequential reprobing of membranes with a variety of antibodies. Stripping and reprobing; the complete removal of primary and secondary antibodies from membranes without antigen damage. Determination of optimum antibody concentration. Figure 1. Principles of ECL Western blotting Oxidised product Secondary Ab-HRP Peracid Light Oxidised form of enzyme + luminol + enhancer Protein Primary Ab Hybond ECL Hyperfilm ECL 6 7

5 CRITICAL PARAMETERS The following points are critical: It is essential to optimise both primary and secondary antibodies for results with high signal and low background due to the extreme sensitivity of the system. It is necessary to work quickly once the membranes have been exposed to the detection system. Wear powder-free gloves when handling film or detection reagents. Do not use azide as a preservative for buffers to be used in immunodetection as it is an inhibitor of horseradish peroxidase. Proper blocking and washing of the membranes is critical for optimum results. It may be necessary to adjust blocking conditions for certain applications. Use as large a volume as possible of washing buffer. Do not allow the membranes to dry out after addition of primary antibody. ADDITIONAL EQUIPMENT AND REAGENTS REQUIRED Equipment Electrophoresis and blotting apparatus (for Western blots) Blotting membrane, recommend Amersham Pharmacia Biotech s Hybond ECL (nitrocellulose) Orbital shaker Forceps with rounded, non-serrated tips X-ray film cassettes, recommend Amersham Pharmacia Biotech s Hypercassette Timer Film, recommend Hyperfilm ECL, film developing facility and reagents Reagents Tris base (tris(hydroxymethyl)aminomethane) Sodium chloride Hydrochloric acid (1M and 5M) Tween 20 Immunodetection reagents (if using RPN 2106 and RPN 2109) Distilled water Disodium hydrogen orthophosphate anhydrous (Na 2 HPO 4 ) Sodium dihydrogen orthophosphate (NaH 2 PO 4.2H 2 0) Buffers and working solutions Buffers 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. 8 9

6 Tris-buffered saline (TBS) ph7.6: 2.42g Tris base (20mM) 8g sodium chloride (137mM) 3.8ml 1M hydrochloric acid Dilute 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 20 concentration in PBS or TBS is suitable for most ECL Western blotting work on nitrocellulose but concentrations carrying from 0.05% to 1% may be required to suit your specific requirements. Biotinylated antibody: It is recommended that the antibody dilution should be optimised to suit different blotting situations. See page 30. The full range of biotinylated antibodies can be found in the current Amersham Pharmacia Biotech catalogue. Storage of working solutions once prepared: All working strength solutions should be stable for one hour at room temperature. For longer periods it is recommended that they be kept in a refrigerator (2-8ºC). For reproducible performance equilibrate to room temperature before use. Storage of buffer once prepared: All buffers should be stable for at least 3 months if prepared in advance and stored at room temperature, although storage in a refrigerator (2-8ºC) may be necessary to avoid microbial spoilage. Sodium azide is not recommended for use as a bacteriocide. Working solutions for ECL immunodetection Membrane blocking agent: Amersham Pharmacia Biotech recommends blocking reagent supplied or substitute with low fat dried milk dissolved in PBS-T or TBS-T; 5g per 100ml (5%). HRP-second antibody It is recommended that the antibody dilution should be optimised to maximise signal and minimise background. If using the second antibodies supplied in RPN 2108, a good starting dilution is 1:1000. See page 31. For details of the recommended ECL HRP antibodies see page

7 12 ECL IMMUNODETECTION PROCEDURE Flow diagram Separate protein sample by electrophoresis Transfer to membrane Block non-specific sites Incubate in primary antibody Incubate in HRPlabelled conjugate ECL detection reagents Expose to film Incubate in biotinylated second antibody Incubate in pre-formed HRP-streptavidin complex ECL detection reagents Expose to film Detailed protocol and notes The protocol outlined on the following pages has been developed in our laboratories to be the optimum for both sensitivity and convenience. A further rapid immunodetection protocol is outlined on page 27 for situations where time is limiting. Users, however, may wish to adapt the protocols to suit their specific needs, and notes and a troubleshooting guide are provided to assist with this. Note: The ECL Western blotting system is extremely sensitive. For results with high signal and low background, it is essential to optimise the concentrations of both primary and secondary antibodies. The high sensitivity means that much higher dilutions of antibodies than used with conventional systems are required. See page 30 for details of optimisation experiment that can be performed to determine the best concentrations of primary and secondary antibodies. During immunodetection, sufficient solution should be used to adquately 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; 4ml of buffer per cm 2 of membrane is suggested. Brief rinses of the membrane before incubating in wash buffer will improve washing efficiency. If exposure times of less than 5 seconds are routinely required it is recommended that the antibodies used are further diluted as it is difficult to perform such exposures. 13

8 14 Protocol Notes 1) Performing electrophoresis and blotting 2) Blocking the membrane Non-specific binding sites are blocked by immersing the membrane in 5% blocking reagent 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 optimum results. Blots may be used immediately or air dried and stored in a desiccator in a refrigerator (2-8ºC) for up to 3 months. 1.2) The Hybond ECL should be pre-wetted in distilled water followed by equilibration in transfer buffer for 5-10 minutes before blotting. 1.3) Stored blots do not require pre-wetting prior to immunodetection. 1.4) Hybond C extra (supported nitrocellulose) may also be used. 1.5) Preliminary results with polyvinyl membranes have given good results, but the system has been optimised for nitrocellulose. 2.1) The combination of Tween and blocking reagent should be suitable for most protein blotting work. Optimum Tween concentrations will vary to suit specific experiments, but a 0.1% Tween 20 (PBS-T) for one hour at room temperature on an orbital shaker. 3) Washing We recommend PBS-T or TBS-T for washing buffer. Briefly rinse the membrane using two changes of washing buffer then wash once for 15 minutes and twice for 5 minutes with fresh changes of the washing buffer at room temperature. concentration in PBS or TBS is suitable for most ECL Western blotting work on nitrocellulose membranes. Certain experimental situations may require alteration of the time and temperature of the blocking incubation. 2.2) Alternative blocking procedures are given in the troubleshooting guide on pages 45 and ) Membranes may be left in the blocking solution overnight in a refrigerator (2-8ºC) if more convenient. 3) As a general rule, as large a volume as possible of washing buffer should be used each time. 15 4) Dilution of primary antibody During the washing step dilute the primary 4) Dilution of the primary antibody required to give optimum results will vary and

9 16 Protocol Notes antibody. (See determination of optimum antibody concentration, p.30) 5) Incubation Incubate the membrane in diluted primary antibody for 1 hour at room temperature. 6) Washing Wash the membrane as detailed in step 3. 7) Dilution During the washing step dilute the biotinylated antibody or the HRP labelled second antibody. If ECL biotinylated markers are to be detected and the protocol is not a biotinstreptavidin system, then streptavidin in HRP conjugate (RPN 1231) at a 1:1500 dilution should be added along with the HRP labelled second antibody. should be determined for each antibody used. These optimisation experiments may be performed by dot blot analysis. 5) Incubation times and temperatures will vary and should be optimised for each antibody. The conditions indicated are recommended starting points. 7) The ECL Western blotting detection reagents can be used with any HRP labelled second antibody or HRP labelled streptavidin and biotinylated antibody. Due to the sensitivity of the ECL Western blotting detection reagents the dilution of the second antibody should be optimised to give the highest signal with minimum background (see p.31). If using the HRP labelled antibody supplied in RPN 2108 a 8) Incubation Incubate the membrane in the diluted second antibody for 1 hour at room temperature. 9) Washing Wash the membrane as detailed in step 3. 10) Incubation If using a biotinylated second antibody, dilute the biotinylated HRP streptavidin complex or streptavidin HRP conjugate and incubate for minutes at room temperature. 11) Washing Wash the membrane 1x15 minutes and 4x5 minutes in fresh changes of wash buffer. suitable starting dilution would be 1: ) Incubation times and temperatures may vary 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 membrane will minimise background. 17

10 18 12) Detection Read through this whole section before proceeding. It is necessary to work quickly once the membranes have been exposed to the detection solution. All steps can be carried out in a dark room; it is only necessary to switch off the light after section Equipment needed are an X-ray film cassette, a roll of SaranWrap (other 'cling-films' may be suitable), a timer and autoradiography film; Hyperfilm ECL (RPN 2103) is recommended. The use of gloves is strongly recommended from this stage onward to prevent hand contact on film, or detection reagents. If possible wear powder-free gloves as the powder can inhibit the ECL detection reagents leading to blank patches on the film Protocol 12.1) Mix an equal volume of detection solution 1 with detection solution 2 to give sufficient to cover the membranes. Notes 12.1) The final volume required is 0.125ml/cm 2 membrane. 12.2) Drain the excess buffer from the washed membranes and place them on a piece of SaranWrap, protein side up. Add the detection reagent to the protein side of the membrane, so that the reagents are held by surface tension on the surface of the membrane. Do not allow the surface of the membranes to become uncovered. 12.3) Incubate for precisely 1 minute at room temperature without agitation. 12.4) Drain off excess detection reagent and wrap membranes in SaranWrap. Gently smooth out air pockets. 12.5) Place the blots, protein side up, in the film cassette. Work as quickly as possible; minimise the delay between incubating the membranes in the detection reagent and exposing them to the film (next step). 12.6) Switch off the lights and carefully place a sheet of autoradiography film such as (Hyperfilm ECL) on top of the membranes, close the cassette and expose for 15 seconds. 12.4) Drain off excess detection reagent by holding the membrane vertically and touching the edge of the membrane against tissue paper. Gently place the membrane, protein side down, on to SaranWrap. Close SaranWrap to form an envelope avoiding pressure on the membrane. 12.5) Ensure that there is no free detection reagent in the film cassette; the film must not get wet. 12.6) Do this in a dark room, using red safelights. Do not move the film whilst it is being exposed ) Remove film, immediately replace with a fresh piece of unexposed film, and 12.7) Develop first piece of film immediately, and on the basis of its

11 20 Protocol reclose film cassette. Notes appearance estimate how long to continue the exposure of the second piece of film. Second exposures can vary from 1 minute to 1 hour; this will depend on the amount of target protein on the membrane. If background is high the membrane may be rewashed twice for 10 minutes with wash buffer and re-detected following steps with slight loss of sensitivity. If overexposure occurs because of high light emission resulting from high target antigen concentration, leave blots in the cassette for 5-10 minutes before re-exposing to film. The ECL detected blots may also be exposed to polaroid film using the ECL mini-camera (RPN 2069), which was specifically designed for blots generated from mini-gel apparatus. The ECL minicamera is suitable for blots up to 52x77mm. 13) Reprobing membranes Following ECL detection it is possible to reprobe the membrane several times to either clarify or confirm results or when small or valuable samples are being analysed (5). Sequential reprobing of membranes with a variety of antibodies is possible following the steps below. The membranes may be stored wet wrapped in SaranWrap in a refrigerator (2-8ºC) after each immunodetection. Protocol Notes 13.1) Wash the membrane for 2x10 minutes in TBS-T or PBS-T at room temperature using large volumes of wash buffer. 13.2) Block the membrane by immersing in 5% blocking reagent in TBS-T or PBS-T for 1 hour at room temperature. 13.2)Refer to note 2.2) on page ) Perform immunodetection as described on pages

12 22 14) Stripping and reprobing membranes The complete removal of primary and secondary antibodies from membranes is possible following the method outlined below. The membranes may be stripped of bound antibodies and reprobed several times. Membranes should be stored wet wrapped in SaranWrap in a refrigerator (2-8ºC) after each immunodetection. Protocol 14.1) Submerge the membrane in stripping buffer (100mM 2-mercaptoethanol, 2% sodium dodecyl sulphate, 62.5mM Tris-HCl ph6.7) and incubate at 50ºC for 30 minutes with occasional agitation. 14.2) Wash the membrane for 2x10 minutes in TBS-T or PBS-T at room temperature using large volumes of wash buffer. 14.3) Block the membrane by immersing in 5% blocking reagent TBS-T or PBS-T for 1 hour at room temperature. Notes 14.1) If more stringent conditions are required the temperature of incubation can be increased up to a maximum of 70 C. 14.2) Membranes may be incubated with the ECL detection reagents and exposed to film to ensure removal of antibodies. 14.3) Refer to note 2.2) on page ) Perform immunodetection as described on pages Summary protocol Prior to immunodetection Step 1) Gel loading 2) Electrophoresis and blotting 3) Preparation for immunodetection Blocking Reagent preparation Prepare samples according to individual requirements. Perform according to usual techniques (6-15). Prepare a 15% solution of blocking reagent in diluent buffer. Immunodetection 23 Notes 1) During immunodetection sufficient dilution should be used to cover the membrane and the container should be agitated gently on some form of mixer platform. 2) All steps for immunodetection can be performed at room temperature.

13 24 Step Incubation time Reagent preparation 1) Block membrane Incubate membrane in solution of blocking reagent 1 hour 2) Wash Rinse membrane twice quickly in fresh wash buffer (see page 10). Wash three times with fresh changes of wash buffer 2 quick rinses 1x15 minutes 2x5 minutes For step 3): Dilute primary antibody in diluent buffer 3) Primary antibody incubation Incubate membrane in diluted primary antibody solution 1 hour 4) Washing Rinse and wash membrane in fresh changes of wash buffer 2 quick rinses 1x15 minutes 2x5 minutes For step 5): Dilute biotinylated antibody or HRP labelled second antibody in diluent buffer 5) Biotinylated antibody or HRPlabelled second antibody incubation Incubate membrane in diluted biotinylated or HRPlabelled second antibody solution minutes For HRP-labelled second antibody omit step 6) and 7) 6) Washing Rinse and wash membrane in fresh changes of wash 2 quick rinses 1x15 minutes 2x5 minutes For step 7): Dilute streptavidinbiotinylated HRP complex or streptavidin HRP conjugate as required 7) Streptavidin-HRP incubation Incubate membrane in diluted complex or conjugate solution minutes 8) Washing Rinse and wash membrane in fresh changes of wash buffer 3 quick rinses 1x15 minutes 2x5 minutes 25

14 26 Step Incubation time Reagent preparation 9) Detection Incubate membrane in equal volumes of detection reagent 1 and detection reagent 2 1 minute 10) Draining 11) Exposure 12) Film developing 13) Interpretation of results 14) Reprobing if required (see page 21) Drain off detection reagent and wrap blots in SaranWrap Put Hyperfilm ECL on top of blot and expose film as required 15 seconds- 60 minutes ADDITIONAL PROCEDURES Rapid immunodetection protocol If time is short the following protocol allows the immunodetection using HRP-labelled antibodies to be completed in just over 2 hours, compared to 4 hours 15 minutes for the standard protocol. If desired, the protocol can be further shortened by also optimising the primary antibody for a shortened incubation. Protocol Notes 27 1) Electrophoresis and blotting. 2) Blocking the membrane Non specific sites are blocked by immersing the membrane in the blocking reagent supplied in phosphate buffered saline Tween (PBS-T) or Tris buffered saline Tween (TBS- T) and incubating for 10 minutes at room temperature with agitation. 3) Dilution of the primary antibody During the blocking stage dilute the primary antibody. 1) See page 14. 2)This protocol has been optimised using the blocking reagent supplied at 10%(w/v). Dried milk (10%) may be used as a substitute. Other blocking agents will need to be tested for their capacity to block effectively in a 10 minute incubation. The short block is suitable for both nitrocellulose and high quality PVDF membranes. 3) The optimum dilution of the primary antibody will vary and should be determined for each antibody used (see page 30).

15 28 Protocol 4) Rinsing Rinse the membrane in PBS-T or TBS-T for 15 seconds, repeat twice. Notes 5) Incubation Incubate the membrane in the diluted primary antibody for 1 hour at room temperature with agitation. 6) Washing We recommend PBS-T or TBS-T for the washing buffer. Briefly rinse the membrane using three changes of wash buffer, then wash twice for 10 minutes with fresh changes of washing buffer, at room temperature. 7) Dilution of the second antibody During the wash steps dilute the HRPlabelled second antibody 5) A further shortening of the immunodetection is possible by increasing the primary antibody concentration, allowing a reduction in the incubation time without compromising sensitivity. However, those wishing to do so must optimise their antibody for the new incubation time. 6) As large a volume of wash buffer as possible should be used at each time. As a guide, there should be at least 4ml per cm 2 of membrane present. 7.1) In order to maintain the same sensitivity as obtained with the standard method the secondary antibody should be used at a higher concentration. As a guideline increasing the concentration by four times should maintain the same sensitivity. However, the high dilution of antibodies normally used for ECL Western blotting, means that such increases in antibody concentration can be performed without requiring excessive amounts of reagents. 7.2) Alternatively, if very short exposures to film are normally performed it may be more convenient to keep the same concentration of secondary antibody and simply increase the exposure time. 29 8) Incubate Incubate the membrane in the diluted primary antibody for 15 minutes at room temperature with agitation. 9) Wash Wash the membrane as described in step 6. 10) Detect Perform the detection with ECL reagents as described on page 18.

16 Determination of optimum antibody concentration Due to the sensitivity of ECL Western blotting, optimisation of antibody concentrations is necessary to ensure the best results. Generally, lower concentrations of both primary and secondary antibodies are required with ECL compared to colorimetric detection. Outlined below are protocols for determining optimal antibody concentrations. 1) Primary antibodies Dot blots are a quick and effective method of determining the optimum dilution of a primary antibody of unknown concentration. Alternatively, a Western blot can be prepared and then cut into several strips. It should be noted that some antibodies may require alternative blocking and washing steps to the ones suggested below. 1.1) Spot a suitable amount of protein sample on to a nitrocellulose membrane such as Hybond ECL, and allow to dry. Prepare one blot for each primary antibody dilution to be tested. 1.2) Incubate in block solution for 1 hour at room temperature with agitation. 1.3) Wash the membranes briefly in two changes of washing buffer then wash once for 15 minutes and twice for 5 minutes with 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 antibody dilution for 1 hour at room temperature ) Wash as detailed in step ) Dilute the secondary antibody and incubate the membranes for 1 hour at room temperature. 1.7) Wash as detailed in step ) Detect using ECL detection reagents as detailed on page 18. The antibody dilution that gives the maximum signal with minimum background should be selected. 2) Secondary antibodies For a secondary antibody of unknown activity, a dot blot is also effective. 2.1) Prepare dot blots and block the membranes as described in 1.1 and ) Incubate in primary antibody for 1 hour at room temperature. 2.3) Wash as detailed in step ) Prepare several dilutions of secondary antibody: eg. 1/1500, 1/3000, 1/5000, 1/10000, 1/ Incubate 1 blot in each antibody dilution for 1 hour at room temperature. 2.5) Wash as detailed in step ) Detect using ECL detection reagents as detailed on page 18. The antibody dilution that gives maximum signal with minimum background should be selected. 31

17 Quantification of proteins on ECL Western blots It has been demonstrated (17) that Hyperfilm ECL exhibits a linear response to the light produced from enhanced chemiluminescence. This relationship can be used for the accurate quantification of proteins of ECL Western blots, using densitometry. The range over which the film response is linear can 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 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 standard dilutions are used. The dilution range should not be greater than one order of magnitude (see example below). It is important that the concentration of the protein to be quantified lies within the standard range. To ensure this, it may be worth running more than one dilution of the protein. 2) If desired, the film to be used can be pre-flashed. This is performed using a modified flash unit such as Sensitize RPN 2051 that has been calibrated (by adjusting its distance from the film), to raise the film optical density 0.1 to 0.2 OD units above that of the standard film. The flash duration should be in the region of 1msec. concentration is only just visible on the film, then the light from the rest of the standards should be in the linear range of the film. 4) The films can then be scanned using a densitometer, and a graph of peak area against protein concentration plotted. The concentration of the protein being quantified can then be read off this graph, taking into account any dilutions made. Example A dilution series of myosin (chicken gizzard) was prepared containing 600ng, 450ng, 300ng, 150ng and 60ng per 10µl of loading buffer. Two further test samples in the range ng were also prepared. Samples were electrophoresed and blotted on to Hybond ECL. Immunodetection was performed using anti-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 film on which the lowest concentration of myosin was just detectable was used for densitometric analysis. The film was scanned using an UltroScan XL laser densitometer (Pharmacia LKB Biotechnology) and a graph was plotted of peak area (OD units) against myosin concentration. The concentrations of the two test samples were then estimated from the standard curve. 3) The Western blot is detected using standard protocols and then exposed to film. For quantification to be accurate, it is important that the light produced is in the linear range of the film. This can be achieved by making several exposures of different lengths of time. If the standard of lowest 32 33

18 Figure 2. ECL detection of myosin standard curve and myosin test samples. Figure 3. Peak area (OD units) against myosin concentration Peak area (OD units) From left to right: myosin standards 600ng, 450ng, 300ng, 150ng, 60ng, myosin test samples 1,2. 15 second exposure to Hyperfilm ECL. Table 1. Peak area (OD units) for myosin standards and test samples. 34 Myosin samples Peak area (OD units) Standards 600ng ng ng ng ng Test samples ng myosin Table 2. Comparison of calculated with actual concentration for the myosin test samples. Test sample Actual Calculated concentration concentration (ng) (ng) 1 240ng ng

19 36 Use of ECL protein molecular weight markers The ECL protein molecular weight markers (RPN 2107) are a mixture of six different proteins labelled with biotin for use in Western blotting following electrophoresis on a polyacrylamide gel prepared by the method of Laemmli (18). Incubation of the blot with streptavidin horseradish peroxidase followed by detection with the ECL Western blotting system will result in a ladder of bands of approximately equal intensity. Protocol 1) Remove 1µl of markers and add to 9µl of gel loading buffer (containing 5% 2-β-mercaptoethanol). 2) Heat to 100ºC for 4 minutes. Samples may be loaded on to the gel immediately, or stored temporarily on ice. 3) Load 10µl per well. Notes 1) Prepare dilution freshly, do not store the markers in loading buffer. 2) Do not subject the markers to more than one denaturation. 3) A 10µl loading is sufficient to produce clearly visible bands after a 15 second exposure using overnight blotting in Towbin buffer (9) and standard ECL Western blotting immunodetection protocols. 4) Following electrophoresis and transfer to nitrocellulose membranes, membranes are processed by standard immunodetection protocols as outlined in the main protocol section. If the protocol used is not a biotinstreptavidin system then streptavidin-hrp (RPN 1231) is added (1:1500) in the final antibody incubation. 4.1) It is strongly advised that milk should not be included in the streptavidin-hrp incubation. The binding of streptavidin to biotin is inhibited in the presence of endogenous biotin in the milk, resulting in a much decreased signal when detected by enhanced chemiluminescence. 4.2) If cross reactivity is observed between the streptavidin-hrp and the protein samples on the blot, it is suggested that the lane containing the markers is removed and incubated in streptavidin-hrp separately. The strip can then be re-aligned with the rest of the membrane for ECL detection. 5) The membranes are then washed and detected using ECL reagents as detailed on page ) The volume of markers required to give optimum results will depend on the 6.1) The loading recommended, will give clearly visible bands after a 15 second

20 38 Protocol electroblotting and immunodetection conditions used and the length of exposure to film required. The exact loading will have to be determined for each application. Notes exposure. If the bands take longer to appear, the probable cause is inefficient transfer to membrane. This is most likely to be a problem with large gels. Transfer should be overnight for tank blotting, and greater than 1 hour for semi-dry blotting. There should be good contact between the gel and the membrane during transfer. For tank blots the use of extra Scotch-brite pads and additional securing of the transfer cassettes, with rubber bands, will improve transfer. 6.2) Conversley, if the bands produced are too intense or a longer exposure would be more convenient, it is suggested that a higher dilution of markers is used. 39 Figure 4. Profile of ECL protein molecular Figure 5. ECL protein molecular weight weight markers. markers calibration line. 1µg sample ECL protein molecular weight markers diluted with 9µl of loading buffer and run on a 12% polyacrylamide gel for 1 hour 1.0 at 150 volts, followed by electroblotting on to 0.9 Hybond ECL overnight at 30 volts. Processing of the blot was outlined in the ECL Western 0.8 blotting protocol, using Streptavidin-HRP 0.7 (RPN 1231,1:1500 dilution) and ECL 0.6 Western blotting detection reagents. The light emission was captured using Hyperfilm ECL 0.5 for a 15 second exposure. Phosphorylase b (97400) Bovine serum albumin (68000) Ovalbumin (46000) Carbonic anhydrase (31000) Trypsin inhibitor (20100) Lysozyme (14400) Electrophoretic mobility Log molecular weight Trailing edge Leading edge

21 40 ADDITIONAL INFORMATION Troubleshooting guide Problem Possible cause Remedy 1) No signal 1.1) No transfer of proteins during Western blotting 1.1.1) Re-evaluate blotting procedure: Stain gels with dye (15) to check transfer efficiency ) Stain membrane with protein stain to check transfer efficiency ) Optimise gel acrylamide concentration, time for transfer and current (if electroblotting) using molecular weight markers covering the molecular weight range expected to be blotted (molecular weight and Stoke s radius both affect transfer), or use a positive control immunoglobulin ) Check that gel and membrane make proper contact during blotting ) Check that gel and membrane are correctly orientated with respect to the anode (15) ) Check that excess temperatures are not reached during electroblotting producing bubbles, or gel/membrane distortion, etc ) Check that antigenicity is not destroyed by treatment for electrophoresis (SDS, urea, boiling, etc) by dot blotting antigen before and after treatment and immunodetect using ECL detection reagents. 1.2) Protein degradation on storage of blots prior to detection 1.2) Use fresh blots ) No retention of proteins on membranes 1.3.1) Assess no transfer of proteins during Western blotting above ) Use fresh supply of membrane to ensure proper hydration.

22 42 Problem Possible cause Remedy 1.4) Detection system 1.4.1) Check the antigen binding capacity of the primary antibody using a dot blot system ) Increase (and optimise) concentration, incubation times, and temperatures of primary antibody ) Low affinity primary antibody: use solutions without Tween ) Increase (and optimise) reagent concentration and incubation times, for the specific application ) Check that the detection reagents are being stored correctly and used as recommended in step 12.1) in the protocol ) Check detection reagents are working: pre-mix small quantities of detection reagent 1 and detection reagent 2 (0.5ml each) and in the dark add 1µl of HRP-labelled antibody. Visible blue light should be produced. 2) Weak signal 2.1) See 1) above 2.2) Insufficient protein loaded on gel 2.3) Low level of signal 2.1) See 1) above 2.2) Load more protein on the gel ) Pre-flashing the film will increase its sensitivity to the signal and linearise its response. This does, however, require care as increased backgrounds may result. Pre-flashing involves hypersensitising the film just before use by pre-exposure to a short flash of light (approximately 1msec). Conventional photographic flash units are suitable when attenuated with a diffuser and Wratten 6B filter, to give a flash of the required intensity to increase the 540nm absorbance of the developed film to 0.15 above that of the unexposed film ) Expose film for an extended period (1-2 hours) ) Poor protein transfer on to membrane. 43 3) Excessive diffuse signal 3.1) Overloading of protein 3.1) Load less protein on gel.

23 44 Problem Possible cause Remedy 4) Uneven/ spotted blot 5) High backgrounds 3.2) Improper gel conditions 3.3) Antibody concentrations too high 4.1) Improper blotting technique 4.2) Unevenly hydrated membrane 4.3) Fingerprints and/or keratin contamination 5.1) Antibody concentrations too high 3.2) Optimise gel, electrophoresis and blotting conditions. Increase acrylamide concentration of gel. Check gel and buffer recipes. Check that no bubbles interfere with transfer from gel to membrane. 3.3) Reduce concentrations of antibodies (see page 30). 4.1) See 1) above ) Use new/fresh membranes ) Make sure that membrane is fully covered and wetted during incubations. 4.3) Avoid touching membrane. Use gloves and blunt forceps. 5.1) Both primary and secondary antibodies will give rise to high background if used at too high a concentration. Antibody optimisation (see page 30) should be performed ) Contaminated blotting equipment 5.3) Contaminated buffers 5.4) Inadequate blocking 5.2) Clean or replace all equipment. 5.3) Ensure all buffers are freshly prepared and filtered ) Check that blocking agent solution has been made up correctly ) Use a freshly prepared solution of blocking agent ) Increase concentration of blocking agent (to 10% at first) ) Include blocking agent in all detection reagent working solutions ) Increase Tween concentration (Caution: Tween may reduce the binding of antibodies, particularly of low affinity primary antibodies) ) Increase incubation time and/or temperature of blocking incubation ) Try alternative blocking agents: 1-10% bovine serum albumin in TBS-T or PBS-T (freshly prepared) % gelatine in TBS-T or PBS-T (freshly prepared).

24 46 Problem Possible cause Remedy 5.5) Problems with membranes Incubation times and temperatures with albumin and gelatine blockers will need to be determined for each case, starting from one hour at room temperature up to overnight at 37ºC or 50ºC, or various combinations thereof (6-13,15). 1% polyvinylpyrrolidone (PVP) in TBS-T or PBS-T ) Check that the membranes are completely immersed in all solutions especially during washing, and that membranes hydrate thoroughly ) Use a fresh supply of membranes ) Use high quality membranes: Hybond ECL (RPN 2020D or RPN 82D) is the recommended nitrocellulose membrane ) Damage to the membrane can cause non-specific binding of the detection reagents. Handle blots carefully with gloved hands and blunt non-serrated forceps. 5.6) Inadequate washing 5.7) Detection reagents 5.8) Over exposure 5.5.5) Use clean forceps to handle blots after washing ) Increase washing times and volumes of wash buffers ) Add Tween to reagents if not included already ) Increase concentration of Tween blocking solution ) Rewash blots twice for 10 minutes in wash buffer and repeat detection steps ) Excess detection reagents in blots. Drain well by absorbing the excess on tissue paper before placing blots in film cassettes ) Expose the film for a minimum period (an initial 15 seconds exposure may be all that is required). If exposure time is too short to be convenient, reduce antibody concentrations (see page 30) ) Leave blots in the cassette for 5-10 minutes before re-exposing to film. 47 Quality control Every batch of ECL detection reagents is functionally tested in a Western blotting application to ensure minimal batch to batch variability.

25 Background and references Principles of ECL detection Luminescence is defined as the emission of light resulting from the dissipation of energy from a substance in an excited state. In chemiluminescence the excitation is effected by a chemical reaction. The chemical reactions of cyclic diacylhydrazides such as luminol have been widely used in chemical analysis (1,2) and extensively studied (3,4). One of the most clearly understood systems is the HRP/hydrogen peroxide catalysed oxidation of luminol in alkaline conditions. Immediately following oxidation, the luminol is in an excited state which then decays to ground state via a light emitting pathway. Enhanced chemiluminescence (2) is achieved by performing the oxidation of luminol by the HRP in the presence of chemical enhancers such as phenols. This has the effect of increasing the light output approximately 1000 fold and extending the time of light emission. The light produced by this enhanced chemiluminescent reaction peaks after 5-20 minutes and decays slowly thereafter with a half life of approximately 60 minutes. The maximum light emission is at a wavelength of 428nm which can be detected by a short exposure to blue-light sensitive autoradiography film for example Hyperfilm ECL. Figure 3. Graph of light emission versus time, showing the difference between chemiluminescence and ECL. Light emission ECL Approx 1h Time Chemiluminescence Figure NH 2 NH 2 1) ISACSSON, U. and WATERMARK, G., Anal. Chim. Acta., 68, pp , ) WHITEHEAD, T. P. et al., Clin. Chem., 25, pp , ) ROSEWELL, D. F. and WHITE, E. H., Methods in Enzymology., 57, Deluca, M. A. (Ed). Academic Press, New York, pp , ) MOTSENBOCKER, M. A., J. Biolum. Chemilum., 2, pp.9-16,

26 5) KAUFMANN, S. H. et al., Anal. Biochem., 161, pp.89-95, ) GERSHONI, J. M. and PALADE, G.E., Anal. Biochem., 131, pp.1-15, ) TOWBIN, H. and GORDON, J., J. Immunol. Meth., 72, pp , ) PELUSO, R.W. and ROSENBERG, G.H., Anal. Biochem., 162, pp , ) TOWBIN, H. STAEHELIN, T. and GORDON, J., Proc. Natl. Acad. Sci. (USA), 76, pp , ) GERSHONI, J. M. and PALADE, G.E., Anal. Biochem., 124, pp , ) BURNETTE, W. M., Anal. Biochem., 112, pp , ) BITTNER, M., KUPFERER, P. and MORRIS, C. F., Anal. Biochem., 102, pp , ) LIN, W. and KASAMATSU, H., Anal. Biochem., 128, pp , ) ANDREWS, A. T., Electrophoresis: Theory, Techniques and Biochemical and Clinical Applications, Second Edition in Monographs on Physical Biochemistry, edited by A.R. Peacock and W. R. Harrington, Oxford Science Publications, ) JOHNSTONE, A. and THORPE, R., Immunochemistry in Practice., Blackwell Science Publications, ) LIFE SCIENCE., 7, pp.12-13, Amersham International plc, ) LAEMMLI, U.K., Nature., 227, pp , ) HOFFMAN, W. L. and JUMP, A. A., Anal. Biochem., 181, pp , Related products Horseradish peroxidase-labelled second antibody conjugates: Mouse Ig, horseradish peroxidase-linked whole NA 931 antibody (from sheep) Rabbit Ig, horseradish peroxidase-linked whole NA 934 antibody (from donkey) Rat Ig, horseradish peroxidase-linked whole NA 932 antibody (from sheep) Human Ig, horseradish peroxidase-linked whole NA 933 antibody (from sheep) Mouse Ig, horseradish peroxidase-linked whole NXA931 antibody (from sheep) general purpose screening reagent Mouse Ig, horseradish peroxidase-linked F(ab ) 2 NA9310 fragment (from sheep) Rabbit Ig, horseradish peroxidase-linked F(ab ) 2 NA9340 fragment (from donkey) Rat Ig, horseradish peroxidase-linked F(ab ) 2 NA9320 fragment (from sheep) Human Ig, horseradish peroxidase-linked F(ab ) 2 NA9330 fragment (from sheep) ECL Plus western blotting reagents ECL blocking agent (40g) RPN2125 ECL PLus western blotting detection reagent pack RPN2124 membrane blocking reagent and HRP labelled anti mouse and anti rabbit antibodies optimized for ECL plus detection ECL Plus western blotting detection reagents RPN2132 (sufficient for 1000cm 2 membrane) ECL Plus western blotting detection reagents RPN2133 (sufficient for 3000cm 2 membrane) 51

27 ECL protein molecular weight markers Six biotinylated proteins, which when incubated RPN 2107 with streptavidin-hrp and ECL Western blotting detection reagents provide a ladder covering molecular weights Sufficient reagents for 25 loadings Streptavidin-horseradish peroxidase RPN 1231 conjugate, 2ml Streptavidin biotinylated HRP complex, 2ml RPN 1051 ECL glycoprotein detection module RPN 2190 Sufficient for 25 8x10cm membrane labellings or 50 solution labellings ECL glycoprotein detection system RPN 2191 Contains: ECL glycoprotein module RPN 2190 ECL Western blotting detection reagents RPN 2209 ECL streptavidin-hrp and blocking reagent RPN 2195 Sufficient for 2000cm 2 membrane ECL protein biotinylation module RPN 2202 Sufficient for 25 8x10cm membrane labellings or 5x2.5mg solution labellings ECL protein biotinylation system RPN 2203 Contains: ECL protein biotinylation module RPN 2202 ECL Western blotting detection reagents RPN 2209 ECL phosphorylation detection system RPN 2221 Contains: ECL phosphorylation module RPN 2220 ECL detection reagents RPN ECL phosphorylation detection module RPN 2220 Sufficient for 2000cm 2 membrane Hybond ECL High quality nitrocellulose membranes, recommended for use with ECL Pack of 10 nitrocellulose membranes, 20x20cm RPN 2020D Pack of 50 nitrocellulose discs, 82mm diameter RPN 82D Hybond-PVDF Recommended for use with ECL Plus Pack of 10 PVDF membranes, 20x20cm RPN 2020P Roll of PVDF embrane, 20x3m RPN 203P Hyperfilm ECL Pack of 25 films, 18x24cm RPN 2103 Pack of 25 films, 30x40 cm RPN 2104 Pack of 25 films, 10x12 inches RPN 1681 Pack of 25 films, 5x7inches RPN 1674 ECL mini-camera RPN 2069 A 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 Hypercassette Hypercassette, 18x24 cm RPN 1642 Hypercassette, 30x40 cm RPN 1644 Hypercassette, 5x7 inches RPN 1648 Amersham Pharmacia Biotech also supplies a full range of ECL products for nucleic acid labelling and detection. For details please contact your local Amersham Pharmacia Biotech office. 53