Amersham™ ECL™ Gel Box - GE Healthcare Life Sciences
Contents
1. WARNING Do not operate the Amersham ECL Gel Box inside a metal tray 14 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 2 Safety 2 1 Safety precautions WARNING Never operate run the Amersham ECL Gel Box without both high voltage plugs connected to the external power supply WARNING High voltage Do not overfill Amersham ECL Gel Box with running buffer Maximum volume is 90 ml per tank WARNING Do not operate Amersham ECL Gel Box close to strong magnetic fields CAUTION Hazardous substances When using hazardous chemicals take all suitable protective measures such as wearing protective glasses and gloves resistant to the substances used Follow local and or national regulations for safe operation CAUTION Do not move Amersham ECL Gel Box during operation gt BPP Pe gt Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 15 2 Safety 2 1 Safety precautions NOTICE To prevent equipment damage only operate Amersham ECL Gel Box in a horizontal position Maintenance WARNING Electrical shock hazard Always switch off the Power supply and disconnect Amersham ECL Gel Box from the Power supply before maintenance and cleaning WARNING Cleaning Do not autoclave bake or microwave Amersham ECL Gel Box WARNING Cleaning Do not wash Amersham ECL Gel Box in a mechanical washer gt PP FP 02. D separation may be required Sample spreading across the gel e Excess salt in the sample e Too much protein applied to the gel Reduce salt by ultrafiltration Optimize the amount of protein applied to the gel Sample contains appreciable carbohydrate Remove the carbohydrate by enzymatic or chemical means Sample contains Use a gradient gel lipoproteins Try addition of a non ionic detergent Protein Excessive heating denaturation and band inversion Start with cold buffer lt 15 C Diffuse protein zones in the gel after staining e SDS still present in the gel e Insufficient reduction of samples Wash the gels extensively 3 x 10 minutes with ultrapure water and use 30 methanol to destain the gel Use 10 TCA to fix the proteins in the gel Add extra reducing agent or change the reducing agent Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC Problems Possible causes 7 Troubleshooting Remedies No green light low current lt 20 mA at start Incomplete circuit Check all power connections Check buffer level in tanks Ensure that the gel cassette is in place Ensure that there is no tape left on the gel cassette Green light but only approx 35 mA at start Leak between tanks with or without gel cassette Check that the tanks are not overfilled Check if the unit is damaged and needs to be replaced Green light but
3. Introduction General information Amersham ECL Gel Box together with Amersham ECL Gel constitute a ready to use mini gel system for high quality separation of proteins Amersham ECL Gel is compatible with Tris Glycine buffer and contains no SDS making it ideal for protein analysis under both native and denaturing conditions Amersham ECL Gel is compatible with standard protein detection protocols and has an extended shelf life for increased convenience Scope of this document These instructions provide information on how to perform polyacrylamide gel electrophoresis PAGE using Amersham ECL Gel Box and Amersham ECL Gel Additionally protocols for post staining and transfer are provided In this chapter This chapter contains the following sections Section See page 1 1 Protein gel electrophoresis overview 6 1 2 Important user information 7 1 3 Regulatory information 9 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 5 1 Introduction 1 1 Protein gel electrophoresis overview 1 1 Protein gel electrophoresis overview Below is a flowchart of protein analysis including separation of proteins using PAGE followed by either Western blotting or post staining detection methods Gel electrophoresis Western blotting detection method Post staining detection method Transfer Post staining Detection Detection 6 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 1 Introduction 1 2 Important u
4. Step Action i Discard the running buffer 2 Rinse thoroughly with distilled water 3 Let the instrument air dry or wipe with a soft damp tissue 4 Keep power cables and connectors dry Store Amersham ECL Gel Box in a dry location at room temperature Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 7 Troubleshooting Problems Possible causes 7 Troubleshooting Remedies Low or no current during the run Interrupted circuit Add some running buffer to each tank of Amersham ECL Gel Box Ensure that the tape is removed from the gel cassette Ensure that the lid is in place Streaking of proteins e Sample overload e Poorly soluble or weakly charged particles such as carbohydrates in sample e High salt concentration in the sample e Contaminants such as membranes or DNA complexes in the sample Load the appropriate amount of protein Centrifuge the samples Change the pH of the sample buffer Heat the sample together with SDS Decrease the salt concentration of the sample solution using dialysis or gel filtration Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 59 Troubleshooting Problems Possible causes Remedies Bands difficult to distinguish Incorrect gel selection sample overloading Select a gel that separates in the desired molecular weight range Reduce loaded protein amount For proteins of similar molecular weight a 2
5. chapter This chapter contains the following sections Section See page 5 1 Electrophoresis protocol 39 5 2 Removing the gel from the cassette 44 5 3 Post staining of the gel 46 5 4 Transfer protocol 53 38 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 5 Protocols 5 1 Electrophoresis protocol 5 1 Electrophoresis protocol In this section This section describes how to perform PAGE using Amersham ECL Gel Box and Amersham ECL Gel Precautions WARNING Do not use Amersham ECL Gel Box if it is not working properly nor if it has suffered any damage for example e damage caused by dropping the equipment e damage by spilling liquid onto it WARNING Do not connect the high voltage cable to an external power supply if it is not working properly nor if it has suffered any damage for example damage to its plug or cable Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 39 5 Protocols 5 1 Electrophoresis protocol Preparations before a run 40 Step Action 1 Prepare 1x running buffer by diluting 19 ml of Amersham ECL Gel Running Buffer 10x in 171 ml water 190 ml buffer is sufficient for one electrophoresis gel 2 Add 90 ml of 1x running buffer to each tank of Amersham ECL Gel Box 3 Cut open the gel package and gently remove Amersham ECL Gel from the package 4 Rinse the gel cassette with distilled water Peel off the tapes from the two legs of the
6. 2012 General Electric Company All rights reserved First published May 2011 All goods and services are sold subject to the terms and conditions of sale of tl he company within GE Healthcare which supplies them A copy of these terms and conditions is available on request Contact your local GE Healthcare representative for the most current information GE Hea thcare Europe GmbH Munzinger Strasse 5 D 79111 Freiburg Germany GE Hea Amersh GE Hea thcare UK Limited am Place Little Chalfont Buckinghamshire HP7 9NA UK thcare Bio Sciences Corp 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 1327 USA GE Hea Sanken thcare Japan Corporation Bldg 3 25 1 Hyakunincho Shinjuku ku Tokyo 169 0073 Japan 28 9943 33 AC 02 2012
7. EAHA MPI m IgE SIH LW EAER DAGRIUEPT RBA AY RENT GA BYE OR AE A AB ANF Ge te 8 mA RNT ED SE EIA A Sah Uy Se Se Se Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 23 2 Safety 2 4 Declaration of Hazardous Substances DoHS List of hazardous substances and their concentrations FRHeSsASMARAANEZHMRES Indication for each major part if substance exceeds limit Meaning 0 Indicates that this toxic or hazardous substance contained in all of the homogeneous materials for this part is below the limit requirement in SJ T11363 2006 RARBASA SVREBBAMAYRMA Heat SJ T11363 2006 EAN A BR BE RAF x Indicates that this toxic or hazardous substance contained in at least one of the homogeneous materials used for this part is above the limit requirement in SJ T11363 2006 e Data listed in the table represents best information available at the time of publication e Applications of hazardous substances in this medical device are required to achieve its intended clinical uses and or to provide better protection to human beings and or to environment due to lack of reasonably eco nomically or technically available substitutes RRBZRSRSWRSSEBRANR HRAAEHS IH HSJ T11363 2006 PRJ EKI REZR ERIRE A A Th AY PE RR AS AY Be ES ATRDATEREKREABTTHSRMARAR BTR b KRSR SWARMS FRIAR DORE AA ERE RP RR 24 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 2 Safety 2 4 Declaration of Hazardous Substanc
8. GE Healthcare Life Sciences Amersham ECL Gel Box User Manual Na OF Na ps ca 5 lt lt Table of Contents Table of Contents t Introductions sse RA 5 1 1 Protein gel electrophoresis Overview uuaceccsccsssssssssssesssssscssssssssssssesssssssssssesssusssees 6 1 2 Important user information 7 13 Regulatory INTOrMOtiON sis sassscnsarnssdesncntiansuaccaaaatiians 9 ESO psn E aa 11 21 Safety PPECCULIONS siiin n 12 2 2 Labels 18 23 Recycling ITO INCHEON csasasssssicascccssevsvectsayasssstacnncousstinasststdladbaansoasvsctooaissctsanaanisigns 21 2 4 Declaration of Hazardous Substances DOHS s s s ss1ssis1ssrssssrssrerssrsestrsere 22 OTD USN eo ees ee 26 31 Amersham ECL Gel Box ucceeccccssssssssssssssssssssssessessssssssssssssssssssssssnsessnnsetsnnnsssnasssssnanenssa 27 3 2 Amersham ECU Gel pgina 30 A MASE IRONY sakcscissansssssssriorsisesscienicuneanicasseaesanoiesuoiouseueiuiomanlanss 34 Te 38 5 1 Electrophoresis protocol occeeececccssssssssssssssssssssssssesesceeseeeeseeceeececeeceecceceeceeseeeeeeseeeesseees 39 5 2 Removing the gel from the Cassette eeeesssssssssscssessunnssesssssseeeeeessssseteees 44 5 3 POSt Staining of the gel u eeeeesssssssescessssesssssecsesseeeeeeesssstuunnnssesseeseeeeeeeeseesetee 46 SA mansien protocole reanna Ra aaa 53 E VOCS IV E pap N 57 PPB SEND puninu eir Naini 59 8 Related ee 62 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 3 1 Introduction I
9. Not needed Example GEHC EPS 301 Without 4 mm rigid insulation inner dis tance gt 6 mm Adapter supplied with Amersham ECL Gel Box gt High voltage power 18 1129 59 supplies with 2 mm Adapter set rigid insulation 4 mm 2 mm for use with 4 mm HV leads to 2 mm power supply output socket ap GEHC EPS power supply with built in sudden load change detection Not compatible EPS 601 EPS 1001 EPS 3501XL Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 4 Installation Mounting the adapter This instruction describes how to mount the adapter delivered with Amer sham ECL Gel Box Step Action a Turn off the power supply and remove the electrical contact from the power supply outlet 2 Insert the adapter in the power supply output socket WARNING Do not use the adapter in 4 mm socket with lt 6 mm internal distance between surface and socket lt 6 mm 3 Tighten the set screw using the 1 5 mm Hex wrench provided with the Amersham ECL Gel Box to lock the expandable plug of the adapter into the socket 4 Make sure that the adapter is locked into the socket Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 37 5 Protocols 5 Protocols About this chapter This chapter presents protocols for PAGE using Amersham ECL Gel Box and Amersham ECL Gel Additionally protocols for post staining and transfer are provided In this
10. RNING Do not operate Amersham ECL Gel Box without the safety lid Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 29 3 System description 3 2 Amersham ECL Gel 3 2 Amersham ECL Gel Introduction This section describes the features of Amersham ECL Gel Design Amersham ECL Gel is designed for PAGE using Amersham ECL Gel Box Gel concentrations Amersham ECL Gels are available as homogenous and gradient gels in the following concentrations e 10 e 12 e 4 12 e 8 16 e 4 20 For information regarding separation ranges for each Amersham ECL Gel see Separation range on page 31 30 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC Separation range 3 System description 3 2 Amersham ECL Gel The following image shows the separation pattern of proteins at different molecular weight for each Amersham ECL Gel 10 12 250 250 130 o 95 aS 72 mm7 55 55 3 6 M28 HE 36 28 17 17 10 4 12 E 250 E 130 Mammo 72 55 36 28 17 8 16 250 130 95 72 55 36 28 7 10 4 20 E 250 E 130 E 95 72 55 E 36 28 17 n 10 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 31 3 System description 3 2 Amersham ECL Gel Gel specifications 32 Parameter Gel matrix Acrylamide bis acrylamide Gel concentration C 2 6 w w T Total concentration w v of acrylamide bisacrylamide is give
11. Related products on page 62 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 53 5 Protocols 5 4 Transfer protocol Preparing the transfer tank Step Action 1 Fill the transfer unit with transfer buffer For information on how to use the transfer unit refer to the instructions provided by the manufacturer of the transfer unit 2 Place the open transfer cassette with the anode side down in a tray with transferbuffer and ensure that it is immersed in at least 3 cm of the transfer buffer 3 Build the stack with the membrane closest to the anode side Below is an illustration of the transfer sandwich O CATHODE Two buffer soaked blotting papers Polyacrylamide Gel UONIAIIP JAJSUDIL Membrane Two buffer soaked blotting papers 6 SSS SS OS SS Se SSeS gt s m 4 Layer 1 Place a 3 mm thick foam sponge in the transfer cassette and press gently to remove any air bubbles 5 Wet the blotting paper in transfer buffer 54 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC Step 5 Protocols 5 4 Transfer protocol Action 10 11 12 I3 14 Layer 2 Place the two pre wetted blotting papers on the sponge and press gently to remove any air bubbles Note Use a pipette or stirring rod to gently roll out any air bubbles trapped while assembling the stack Layer 3 Place the pre wetted membrane on layer 2 and remove all air bubbles Equilibrate the gel in cold transfer
12. Remove the Staining solution Rinse three times in distilled water for one minute each time Developing Add developing solution and gently shake for 3 minutes Transfer the gel to stopping solution when the bands spots have reached desired intensity Note If the background and and the bands are too strong use a cold transfer buffer and or transfer the gel to stopping solution before desired density has been reached Stopping Gently shake the gel in stopping solution for 120 minutes Preserving Add preserving solution and shake gently for 120 minutes Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 5 Protocols 5 4 Transfer protocol 5 4 Transfer protocol In this section This section describes how to transfer the proteins from the gel to a blotting membrane using tank transfer and semi dry blotting Tank transfer Transfer buffer Tris glycine buffer 25 mM Tris 192 mM glycine 20 methanol Prepare fresh Note Use a pre chilled transfer buffer Preparing the membrane Step Action 1 Pre wet the membranes e NitroCellulose NC membrane Pre wet the membranes in distilled water for five minutes followed by 10 minutes in transfer buffer e PVDF membrane Pre wet the PVDF membranes first in methanol for 20 seconds then in distilled water for 5 minutes and finally in cold transfer buffer for at least 10 minutes Note Use a pre cut membrane provided by GE Healthcare See Chapter 8
13. WARNING Cleaning Do not use abrasive creams or scourers 16 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 2 Safety 2 1 Safety precautions NOTICE Cleaning Clean the instrument with distilled water and wipe dry with a soft damp tissue Let the instrument dry completely before use Materials Safety Data Sheet It is recommended to read the Materials Safety Data Sheet MSDS before using Amersham ECL Gel Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 17 2 Safety 2 2 Labels 22 Labels Introduction This section describes the safety labels and labels concerning hazardous substances that are attached to the Amersham ECL Gel Box instrument Labels on Amersham ECL Gel Box The illustration below shows an example of the label that is attached to the bottom of the Amersham ECL Gel Box Amersham ECL Gel Box Code no 28 9906 08 Max Voltage 200V Serial no 1234570 Max Power 20W AACEX K VEEN ZB Go Protection Class 0 Made in ISRAEL Mfg for GE Healthcare Bio Sciences AB Mfg by Gene Bio Application Ltd 5 1 6 Safety symbols The following safety symbols are used in the label Meaning Warning Read the user documentation before using the instrument Do not open any covers or replace parts unless specifically stated in the user documentation 18 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 2 Safety 2 2 Labels Mea
14. approx 65 mA at start Leak between tanks with gel cassette Check that tanks are not overfilled Check if the unit is damaged and needs to be replaced Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 61 8 Related products 8 Related products In this chapter This chapter presents a subset of related products For more information refer to www gelifesciences com Sample preparation Product Quantity Code no SDS PAGE Clean up kit 50 samples 80 6484 70 Mammalian Protein Extraction Buffer 1 for 500 ml 28 9412 79 2 D Quant Kit 500 assays 80 6483 56 Amersham markers Product Quantity Code no Low Range Rainbow Molecular Weight Markers 250 ul RPN755E High Range Rainbow Molecular Weight Markers 250 ul RPN756E Full Range Rainbow Molecular Weight Markers 250 ul RPN800E Amersham ECL DualVue Western Blotting Markers 1 pack RPN810 25 loadings Amersham ECL Plex Fluorescent Rainbow Markers 120 ul RPN850E Amersham ECL Plex Fluorescent Rainbow Markers 500 ul RPN851E 62 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC Power supplies Product Quantity 8 Related products EPS 301 Power Supply 18 1130 01 Amersham ECL Gel Product Quantity Code no Amersham ECL Gel 10 10 wells 10 28 9898 04 Amersham ECL Gel 12 10 wells 10 28 9898 05 Amersham ECL Ge
15. ated before decommissioning and all local regulations shall be followed with regard to scrapping of the equipment Disposal general instructions When taking Amersham ECL Gel Box out of service the different materials must be separated and recycled according to national and local environmental regulations Disposal of electrical components Waste of electrical and electronic equipment must not be disposed as unsorted municipal waste and must be collected separately Please contact an authorized representative of the manufacturer for information concerning the decommissioning of equipment H Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 21 2 Safety 2 4 Declaration of Hazardous Substances DoHS 2 4 Declaration of Hazardous Substances DoHS Introduction The following product pollution control information is provided according to J T11364 2006 Marking for Control of Pollution caused by Electronic Information Products RYESJ T11364 2006 BFS ORAS SRAMTA KK RAHSA Symbols used in pollution control label AFEA mR aM Si Meaning This symbol indicates the product contains hazardous Oo materials in excess of the limits established by the Chinese A standard SJ T11363 2006 Requirements for Concentration Limits for Certain Hazardous Substances in Electronic Information Products The number in the symbol is the Environment friendly Use Period EFUP which indicates the period during which the toxic
16. bit IgG HRP Linked Whole Ab from donkey 1ml NA934 1ML ECL Mouse IgG HRP Linked F ab fragment from sheep 1ml NA9310 1ML ECL Rabbit IgG HRP Linked F ab fragment from 1ml NA9340 1ML donkey ECL Plex goat a mouse lIgG Cy2 150 ug 28 9011 08 ECL Plex goat a rabbit IgG Cy2 150 ug 28 9011 10 ECL Plex goat a rabbit IgG Cy3 150 ug 28 9011 06 ECL Plex goat a mouse IgG Cy3 150 ug PA43009 ECL Plex goat a rabbit IgG Cy5 150 ug PA45011 ECL Plex goat a mouse IgG Cy5 150 ug PA45010 Detection reagents Product Quantity Amersham ECL Select Western blotting reagents for 1000 cm RPN2235 membrane Amersham ECL Prime Western blotting reagents for 1000 cm RPN2232 membrane Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 67 8 Related products Product Quantity Amersham ECL Plex Amersham ECL Western blotting reagents for 1000 cm RPN2109 membrane Amersham ECL Western blotting reagents for 4000 cm RPN2106 membrane Amersham ECL Western blotting reagents for 6000cm RPN2134 membrane CyDye conjugated Antibodies Product Quantity Amersham ECL Plex Western Blotting Combination Pack 1 RPN998 Cy3 Cy5 Hybond ECL Amersham ECL Plex Western Blotting Combination Pack 1 RPN999 Cy3 Cy5 Hybond LFP for two slab gels Autoradiography Films Product Quantity Amersham Hyperfilm ECL 5 x 7 inches 50 sheets 28 9068 35 A
17. buffer for 10 to 20 minutes Layer 4 Place the gel on the membrane and ensure there are no air bubbles Layer 5 Cover the gel with two pre wetted sheets of blotting paper Layer 6 Finally place a sponge of 3 mm and again press gently to remove any air bubbles Close the cassette and press lightly to lock the tabs Place the cassette into the tank Run the transfer according to the recommendations from the transfer tank supplier Recommended running conditions for transfer Parameter Voltage 25 V maximum 400 mA Temperature 4 C Time 2 5 hours with constant stirring Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 55 5 Protocols 5 4 Transfer protocol Semi dry transfer Introduction In semi dry transfer the gel is placed in direct contact with the membrane and several layers of filter papers soaked in transfer buffer are placed above and below the gel and the membrane e The filter papers gel and membrane are sandwiched between two plates that form an anode and a cathode when an electric field is applied e The filter papers and the membrane should be carefully cut to be a few millimeters smaller than the length and the width of the gels This ensures that the filter papers and membranes do not overlap the gel forming a potential short cut for the current leading to inefficient or uneven transfer of proteins e Ensure that no air bubbles form when applying the gel to the memb
18. cassette 5 Place Amersham ECL Gel in Amersham ECL Gel Box so that the well side of the cassette faces toward the cathode and the other cassette leg faces toward the anode 6 Place the safety lid on top of Amersham ECL Gel Box 7 Connect Amersham ECL Gel Box to the power supply EPS 301 and pre run the gel for 12 minutes at 160 V WARNING Do not exceed the maximum operating voltage of 200 V Note Expected start current approx 45 mA 8 Once the pre run is finished switch off the power 9 Remove the safety lid Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 5 Protocols 5 1 Electrophoresis protocol Step Action 10 Wiggle the comb back and forth and bring it straight up from the cassette to make the wells available for sample loading Note Small gel pieces can be detached from the well container This will not affect gel performance Note Do not discard the comb 11 Add 6 ml of 1x running buffer to the well container Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 41 5 Protocols 5 1 Electrophoresis protocol Sample loading To load the samples follow the instructions Step Action il Prepare the samples by adding sample and 2x sample buffer in a 1 1 mixture Example for 10 ul sample add 10 ul 2x sample buffer Note For native conditions use a sample buffer without SDS and DTT Heat the samples at 95 C for 5 minutes Spin down the samples quickly i
19. ch and power cord Do not block access to the Power switch on the Power supply and the Power cord The Power switch must always be easy to access The Power cord must always be easy to disconnect CAUTION During electrophoresis very low quantities of gases are produced at the electrodes Make sure that Amersham ECL Gel Box is run in a well ventilated area CAUTION Amersham ECL Gel Box is designed for indoor use only 34 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 4 Installation Connecting GE Healthcare power supplies Connect Amersham ECL Gel Box to the power supply according to the instructions supplied with the power supply Connecting other power supplies The power supply must have a rigid insulating sleeve output connector that fulfills the requirements for 200 V DC for example EPS 301 If a power supply with 4 mm deep socket gt 6 mm internal distance between surface and socket is used it is recommended to use the adapter delivered with Amersham ECL Gel Box For mounting instructions see Mounting the adapter on page 37 Note Amersham ECL Gel Box is delivered with two adapters identical apart from color one black and one red shown above Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 35 4 Installation Power supply compatibility guide for Amersham ECL Gel Box Power supply type Equipped with 4 mm rigid insulation Adapter
20. ect 12 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 2 Safety 2 1 Safety precautions CAUTION During electrophoresis very low quantities of gases are produced at the electrodes Make sure that Amersham ECL Gel Box is run in a well ventilated area CAUTION Amersham ECL Gel Box is designed for indoor use only Operation WARNING High voltage Before use check that Amersham ECL Gel Box is completely dry on the outside before connecting it to a power supply Wipe dry with a cloth WARNING Do not use Amersham ECL Gel Box if it is not working properly nor if it has suffered any damage for example gt gt FE gt e damage caused by dropping the equipment e damage by spilling liquid onto it Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 13 2 Safety 2 1 Safety precautions WARNING High voltage Do not connect the high voltage cable to an external power supply if it is not working properly nor if it has suffered any damage for example damage to its plug or cable WARNING Electrical shock hazard Always switch off the power supply and disconnect Amersham ECL Gel Box from the power supply before removing the safety lid WARNING Do not operate Amersham ECL Gel Box without a full functioning safety lid in place WARNING Do not exceed the maximum operating voltage of 200 V gt gt gt Pm gt
21. ed in the table below by fulfilling the corresponding harmonized standards A copy of the Declaration of Conformity is available on request Directive 2006 95 EC Low Voltage Directive LVD 2004 108 EC Electromagnetic Compatibility EMC Directive CE Marking cE The CE marking and the corresponding declaration of conformity is valid for the instrument when it is e used as a stand alone unit or e connected to other CE marked GE Healthcare instruments or e connected to other products recommended or described in the user documentation and e used in the same state as it was delivered from GE Healthcare except for alterations described in the user documentation Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 9 1 Introduction 1 3 Regulatory information International standards This product fulfills the requirements of the following standards Standard Description control and laboratory use EN 61010 1 IEC Safety requirements for Harmonized 61010 1 UL61010 electrical equipment for with 1 CAN CSA C22 2 measurement control and 2006 95 EC No 61010 1 laboratory use EN 61010 031 Safety requirements for hand Harmonized IEC 61010 031 held probe assemblies for with electrical measurement and test 2006 95 EC EN 61326 1 EMC emissions and immunity Harmonized requirements for electrical with equipment for measurement 2004 108 EC Regulatory c
22. es DoHS List of hazardous substances Component Hazardous substance name ABSASYVARTR BE E Ph Hg Cd Cr6 PBB PBDE KM BRK SRI Amersham 0 0 0 0 0 0 ECL Gel Box 28 9902 521 1 The product has not been tested as per the Chinese standard SJ T11363 2006 Requirements for Concentration Limits for Certain Hazardous Substances in Electronic Information Product Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 25 3 System description 3 System description About this chapter This chapter describes the features of Amersham ECL Gel Box and Amersham ECL Gel In this chapter 26 This chapter contains the following sections Section See page 3 1 Amersham ECL Gel Box 27 3 2 Amersham ECL Gel 30 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 3 1 Amersham ECL Gel Box Introduction 3 System description 3 1 Amersham ECL Gel Box This section describes the features of Amersham ECL Gel Box Overview The illustration below shows the main parts of Amersham ECL Gel Box Description Description the plastic case 1 Power cord to external Cathode located under power supply neath the plastic case 2 Anode located underneath Buffer tanks for running buffer 3 Indicator light Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 27 3 System description 3 1 Amersham ECL Gel Box Gel Box specifications Parameter Value Dimen
23. l 4 12 10 wells 10 28 9898 06 Amersham ECL Gel 8 16 10 wells 10 28 9898 07 Amersham ECL Gel 4 20 10 wells 10 28 9901 54 Amersham ECL Gel 10 15 wells 10 28 9901 55 Amersham ECL Gel 12 15 wells 10 28 9901 56 Amersham ECL Gel 4 12 15 wells 10 28 9901 57 Amersham ECL Gel 8 16 15 wells 10 28 9901 58 Amersham ECL Gel 4 20 15 wells 10 28 9901 59 Amersham ECL Gel 10 2 wells 10 28 9901 60 Amersham ECL Gel 12 2 wells 10 28 9901 61 Amersham ECL Gel 4 12 2 wells 10 28 9901 62 Amersham ECL Gel 8 16 2 wells 10 28 9901 63 Amersham ECL Gel 4 20 2 wells 10 28 9901 64 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 63 8 Related products Product Quantity Amersham ECL Gel 10 10 wells 2 28 9898 08 Amersham ECL Gel 12 10 wells 2 28 9898 09 Amersham ECL Gel 4 12 10 wells 2 28 9901 51 Amersham ECL Gel 8 16 10 wells 2 28 9901 52 Amersham ECL Gel 4 20 10 wells 2 28 9901 53 Electrophoresis buffers Product Quantity Amersham ECL Gel Running Buffer 10x 2x 125ml 28 9902 52 250 mM Tris HCI 1 92 M glycine 1 SDS Bromophenol Blue 10g 17 1329 01 Gel staining reagents Product Quantity Code no Deep Purple Total Protein Stain 5ml RPN6305 25ml RPN6306 PlusOne Silver Staining Kit Protein 1 17 1150 01 PlusOne Coomassie Blue PhastGel R 350 Tablets 40 Tablets 17 0518 01 64 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC Transfe
24. mersham Hyperfilm ECL 18 x 24 cm 50 sheets 28 9068 36 68 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 8 Related products Imaging Systems Product Quantity Typhoon Variable Mode Imagers Typhoon FLA 9500 1 29 0040 80 Typhoon FLA 7000 1 28 9558 09 ImageQuant Imagers ImageQuant LAS 4000 1 28 9558 10 ImageQuant LAS 4010 1 28 9558 11 ImageQuant LAS 4000 mini 1 28 9558 13 ImageQuant LAS 500 I 29 0050 63 Software and Accessories Product Code no CD and getting started ImageQuant TL 7 0 and ImageQuant TL SecurlTy 8 0 Software package 28 9380 94 with Getting Started Guide Licenses for ImageQuant TL only ImageQuant TL single user license 28 9236 62 Licenses for ImageQuant TL v7 01 and IQTL SecurlITy v8 0 ImageQuant TL 7 01 and ImageQuant TL SecurlTy 8 0 single user license 28 9332 73 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 69 For local office contact information visit www gelifesciences com contact GE Healthcare Bio Sciences AB Bj rkgatan 30 751 84 Uppsala Sweden www gelifesciences com eclgel imagination at work GE imagination at work and GE monogram are trademarks of General Electric Amershi Company am Deep Purple ECL ECL DualVue ECL Plex Hybond Hyperfilm ImageQuant PhastGel PlusOne Protran Rainbow and Typhoon are trademarks of GE Healthcare companies 2011
25. n Heat to 60 C stirring constantly and filter before use Washing Distilled water solution Destaining 1 Mix 250 ml ethanol and 80 ml acetic acid solution 2 Make up to 1 01 with distilled water 46 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 5 Protocols 5 3 Post staining of the gel Solution Preparation Preserving 1 Mix 25 ml of 87 v v glycerol with distilled water solution 2 Make up to 250 ml with destaining solution Coomassie staining protocol Step Action 1 Fixation Immediately after electrophoresis immerse the gel in fixing solution for 30 minutes This solution precipitates the proteins and allows the SDS to diffuse out of the gel 2 Staining Discard the fixing solution Stain the gel for 10 minutes in staining solution Cover the staining dish 3 Washing Rinse the gel once in distilled water 4 Destaining Destain the gel by changing the destaining solution several times until the stained protein bands are clearly visible against the clear background 5 Preserving Soak the destained gel in preserving solution for 30 minutes Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 47 5 Protocols 5 3 Post staining of the gel Deep purple staining Solution Introduction This section presents a Deep Purple staining protocol recommended by GE Healthcare For more information refer to RPN6305 Deep Purple Total Protein Stain Deep Purple staining sol
26. n a microcentrifuge and load the samples directly into the wells in the gel Note A maximum of 0 5 ug band per well of sample can be loaded Overloading may cause smearing and distortion Note To ensure uniform mobility load an equal volume of 1x SDS sample buffer into any unused well Place the safety lid on top of Amersham ECL Gel Box Running conditions Run the gel according to the recommendations below Parameter Voltage 160 V Expected current Start approx 45 mA End 23 to 33 mA 42 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 5 Protocols 5 1 Electrophoresis protocol Parameter Run time 60 minutes WARNING Do not exceed the maximum operating voltage of 200 V Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 43 5 Protocols 5 2 Removing the gel from the cassette 5 2 Removing the gel from the cassette In this section This section describes how to remove the gel from the Amersham ECL Gel Box cassette after an electrophoresis run Removing the gel from the cassette To remove the gel do the following Step Action 1 Once the run is completed shut off the power disconnect the electrodes remove the safety lid and finally remove the gel cassette from Amersham ECL Gel Box 2 Open the cassette by inserting the edge of the comb in the slot opposite the sample wells and twist 3 Remove the top plate from the gel cassette a
27. n as percentage in the respective Amersham ECL Gel product name Gel thickness 1 4mm Gel size 7 5 cm x 8 0 cm W x H Sample well configuration 2 10 and 15 well gels are available Sample well volume 2 wells 100 ul 10 wells 35 ul 15 wells 20 ul Stacking gel 4 Buffer system in gel Tris HCl Running buffer e Denatured conditions e Native conditions e 25 mM Tris 192 mM glycine 0 1 SDS pH 8 3 e 25 mM Tris 192 mM glycine pH 8 3 Sample buffer e g Tris HCI SDS or any other buffer suitable for the application Shelf life 12 months from date of manufacture Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC Buffer solutions Solution 3 System description 3 2 Amersham ECL Gel Preparations Running buffer 1x 2x SDS Sample 1 Mix 2 5 ml of 0 5 M Tris HCI pH 6 8 4 0 ml of buffer 10 w v SDS 2 0 ml of glycerol 2 0 mg of bromphenol blue and 310 mg of DTT 2 Make up to 10 ml with distilled water Amersham ECLGel Amersham ECL Gel Running Buffer 10x contains 250 mM Tris HCl 1 92 M glycine 1 SDS Prepare 1x buffer for denatured conditions by adding distilled water in a 1 10 dilution Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 33 4 Installation 4 Installation In this chapter This chapter describes how to connect Amersham ECL Gel Box to a power supply Precautions WARNING Access to power swit
28. nd allow the gel to sit on the bottom plate 44 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 5 Protocols 5 2 Removing the gel from the cassette Step Action 4 Cut the stacking gel with the end of the top plate approximately 2 cm downstream of the wells Repeat the procedure on the other side of the gel to remove the front i 5 Hold the gel cassette bottom plate over a container with suitable buffer with the gel facing downwards Gently push a tweezer between the gel and the cassette bottom plate until the gel is removed from the bottom plate Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 45 5 Protocols 5 3 Post staining of the gel 5 3 Post staining of the gel In this section All standard staining procedures including Coomassie staining Silver staining and Deep Purple staining may be used with Amersham ECL Gel For staining solutions recommended by GE Healthcare see Gel staining reagents on page 64 Coomassie staining Introduction This section presents a Coomassie staining protocol recommended by GE Healthcare Coomassie staining solutions 250 ml of each solution is used in each step Solution Preparation Fixing 1 Mix 400 ml of ethanol and 100 ml of acetic acid with solution distilled water 2 Make up to 1 01 with distilled water Staining 1 Mix 1 tablet of PhastGel Blue R 350 with destaining solution solution 2 Make up to 400 ml with destaining solutio
29. ning Warning High Voltage Always make sure that the instrument is disconnected from electric power before opening the lid or disconnecting any electric equipment The instrument complies with the requirements for electromagnetic compliance EMC in Australia and New Zealand N3732 The instrument complies with applicable European C directives The instrument is listed according to T V Rheinland The instrument is protected throughout by double E insulation Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 19 2 Safety 2 2 Labels Labels concerning hazardous substances 20 The following symbols on the labels concern hazardous substances Meaning This symbol indicates that electrical and electronic equipment must not be disposed of as unsorted municipal waste and must be collected separately Please contact an authorized representative of the manufacturer for information concerning the decommissioning of equipment 20 X This symbol indicates that the product contains hazardous materials in excess of the limits established by the Chinese standard SJ T11363 2006 Requirements for Concentration Limits for Certain Hazardous Substances in Electronic Information Product Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 2 Safety 2 3 Recycling information 2 3 Recycling information Decontamination Amersham ECL Gel Box shall be decontamin
30. ompliance of connected equipment Any equipment connected to Amersham ECL Gel Box should meet the safety requirements of EN 61010 1 IEC 61010 1 or other relevant harmonized standards Within EU connected equipment must be CE marked 10 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 2 Safety About this chapter 2 Safety This chapter presents safety precautions for Amersham ECL Gel Box Labels on the instrument and information regarding recycling are also included in this chapter In this chapter This chapter contains the following sections Section See page 2 1 Safety precautions 12 2 2 Labels 18 2 3 Recycling information 21 2 4 Declaration of Hazardous Substances DoHS 22 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 11 2 Safety 2 1 Safety precautions 2 1 Safety precautions General precautions WARNING Do not use Amersham ECL Gel Box in any other way than described in the Amersham ECL Gel Box Instructions CAUTION Operation of Amersham ECL Gel Box should be performed by properly trained personnel only CAUTION Do not use any accessories not supplied or recommended by GE Healthcare Installation gt Pe Pp WARNING Access to power switch and power cord Do not block access to the Power switch on the Power supply and the Power cord The Power switch must always be easy to access The Power cord must always be easy to disconn
31. or hazardous substances or elements contained in electronic information products will not leak or mutate under normal operating conditions so that the use of such electronic information products will not result in any severe environmental pollution any bodily injury or damage to any assets The unit of the period is Year In order to maintain the declared EFUP the product shall be operated normally according to the instructions and environmental conditions as defined in the product manual and periodic maintenance schedules specified in Product Maintenance Procedures shall be followed strictly 22 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 2 Safety 2 4 Declaration of Hazardous Substances DoHS Meaning Consumobles or certain parts may have their own label with an EFUP value less than the product Periodic replacement of those consumables or parts to maintain the declared EFUP shall be done in accordance with the Product Maintenance Procedures This product must not be disposed of as unsorted municipal waste and must be collected separately and handled properly after decommissioning Bie RBA me A HIISI 711363 2006 HF BE RhSSeaSsynhReEk PREVARAMA PRA PAYA A a RE RAR FP ELE BEAWAREL BSBASMRATAREAHMRE A PARAS mo FRAMERS SARWRAR W PER SASH BUA HRUE FB BRAN SER EF BBR WHR m A LE DBRAMAAHKTE SER ARET RAS AFR BE BY BES A RE ER P mP BRE ER He SDE RE SA eR eA Rink FARO RE ARIA ALE TE eae IMR
32. r equipment Product 8 Related products TE 70 PWR Semi Dry Transfer Unit 14 x 16 cm 11 0013 41 TE 77 PWR Semi Dry Transfer Unit 21 x 26 cm 11 0013 42 Amersham ECL Multiprobe 11 0033 95 Amersham ECL Multiprobe XL 11 0033 96 TE 22 Mini Tank Transfer Unit 80 6204 26 TE 62 Transfer Unit 80 6209 58 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 65 8 Related products Blotting membranes Product Quantity Blotting paper 3 MM Chr 20 x 20 cm 100 sheets 3030 861 Blotting sandwich Protran BA83 0 2 um blotting sandwich 10 units 28 9909 83 8 x 7 5 cm Protran BA85 0 45 um blotting sandwich 10 units 28 9909 93 8 x 7 5 cm Membranes Hybond ECL 20 x 20 cm 10 sheets RPN2020D Hybond ECL 8 x 7 5 cm 10 sheets RPN7 58D Hybond LFP 20 x 20 cm 10 sheets RPN2020LFP Hybond LFP 8 x 7 5 cm 10 sheets 28 9909 84 Hybond P 20 x 20 cm 10 sheets RPN2020F Hybond P 8 x 7 5 cm 10 sheets 28 9909 83 Amersham Blocking agents Product Quantity Code no ECL Prime Blocking Agent 40g RPN418 ECL Blocking Agent 40g RPN2125 66 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC Amersham ECL HRP linked Secondary 8 Related products Antibodies Product Quantity ECL Mouse IgG HRP Linked Whole Ab from sheep 1ml NA931 1ML ECL Human IgG HRP Linked Whole Ab from sheep 1ml NA933 1ML ECL Rab
33. rane Bubbles will cause blank spots on the membrane where no protein transfer occurs e Prolonged semi dry blotting may lead to overheating and gel drying due to buffer depletion Semi dry transfer works well for most proteins but may be less efficient for large proteins Protocol information Please refer to 28 4025 91 for detailed instructions for use of Semi dry blotters TE70 and TE77 Post transfer After transfer continue with Western blotting detection or Post staining detection For information regarding GE Healthcare recommended products see Chapter 8 Related products on page 62 56 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 6 Maintenance 6 Maintenance Precautions WARNING Electrical shock hazard Always switch off the power supply and disconnect Amersham ECL Gel Box from the power supply before maintenance and cleaning WARNING Cleaning Do not autoclave bake or microwave Amersham ECL Gel Box WARNING Cleaning Do not wash Amersham ECL Gel Box in a mechanical washer WARNING Cleaning Do not use abrasive creams or scourers NOTICE Cleaning Clean the instrument with distilled water and wipe dry with a soft damp tissue Let the instrument dry completely before use OP gt gt gt Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 57 6 Maintenance Cleaning Storage 58 To clean Amersham ECL Gel Box do as follows
34. room temperature covered from light using the prepared working staining solution Prior to diluting with borate buffer ensure that the Deep Purple concentrate is at room temperature 3 Washing Wash for 90 minutes in washing solution 4 Acidifying Acidify gels for 45 minutes Further washes in this solution can be performed if desired 5 Imaging Imaging is best performed using laser scanning fluorescence flat bed imaging systems such as the GE Typhoon FLA laser scanners Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 49 5 Protocols 5 3 Post staining of the gel Step Action 6 Storing After imaging the gels can be stored in the dark at 8 C for several weeks in storage solution pH 2 3 Note This allows for further imaging at a later date if required For long term storage up to 6 months add Deep Purple stain in 1 200 dilution Silver staining 50 Introduction This section presents a Silver staining protocol recommended by GE Healthcare Refer to Silver Staining Kit Protein 71 7177 00 for more information Silver staining solutions Solutions Preparations Fixing 1 Mix 75 mlethanol and 25 ml glacial acetic acid solution 2 Make up to 250 ml with distilled water Sensitizing 1 Mix 75 ml ethanol 10 ml sodium thiosulfate 5 w v solution and 1 packet of sodium acetate 17 g 2 Make up to to 250 ml with distilled water Before use Add 1 25 ml glutaraldehyde 25
35. ser information 1 2 Important user information Intended use Amersham ECL Gel Box and Amersham ECL Gel are intended for PAGE Amersham ECL Gel Box and Amersham ECL Gel are intended for research use only and shall not be used in any clinical procedures or for diagnostic purposes Safety notices This user documentation contains WARNINGS CAUTIONS and NOTICES concerning the safe use of Amersham ECL Gel Box See definitions below Warnings WARNING WARNING indicates a hazardous situation which if not avoided could result in death or serious injury It is important not to proceed until all stated conditions are met and clearly understood Cautions CAUTION CAUTION indicates a hazardous situation which if not avoided could result in minor or moderate injury Itis important not to proceed until all stated conditions are met and clearly understood Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC T 1 Introduction 1 2 Important user information Notices NOTICE NOTICE indicates instructions that must be followed to avoid damage to the product or other equipment 8 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 1 3 Regulatory information Introduction 1 Introduction 1 3 Regulatory information This section describes the directives and standards that are fulfilled by Amersham ECL Gel Box CE Conformity This product complies with the European directives list
36. sions Gel Box 167 x 148 x 43 5 mm W x H x D Gel cassette 116 8 x 84 x 15 6 mm W x H xD Maximum voltage 200 V Operating temperature 4 C to 40 C Recommended power supply EPS 301 Maximum power 20 W Maximum current 100 mA Degree of protection IPX5 ECL Gel cassette The image below shows the Amersham ECL Gel cassette Description Description 1 Comb 3 Cassette 2 Well container Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 3 System description 3 1 Amersham ECL Gel Box Power supply Amersham ECL Gel Box is powered by an external power supply EPS 301 See Power supplies on page 63 for more information If other power supply with a 4mm deep socket gt 6 mm internal distance between surface and socket is used it is recommended to use the adapter delivered with Amersham ECL Gel Box See Connecting other power supplies on page 35 for more information Safety lid Amersham ECL Gel Box is equipped with a safety lid which shall be placed on top of the base unit before a power supply is connected ae p aa S The safety lid has two magnets which will activate current only when their magnetic fields are in contact with the electrodes inside the base unit of Amersham ECL Gel Box This means that Amersham ECL Gel Box is only powered when the safety lid is in the correct position Removing the safety id will switch off the high voltage to the electrodes WA
37. utions Preparation staining solution Fixating 1 Mix 150 ml ethanol and 10 g citric acid approx pH 2 3 Acidifying solution 2 Make up to 1 01 with distilled water Note Fixing solution can be stored at room temperature for up to six months Working 1 Disolve 6 2 g boric acid in 800 ml distilled water Adjust to pH 10 5 with NaOH 2 Make up to 1 01 with distilled water Note Boric acid sodium hydroxide buffer can be stored at room temperature for up to six months Note Staining solution should be made fresh just before use by adding 1 part Deep Purple to 200 parts borate buffer for gels Prior to diluting with borate buffer ensure that the Deep Purple concentrate is at room temperature 48 Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC Solution 5 Protocols 5 3 Post staining of the gel Preparation Washing solution 1 Mix 150 ml ethanol with distilled water 2 Make up to 1 01 with distilled water Note Washing solution can be stored at room temperature for up to six months Storing solution 1 Add 10 g citric acid to 500 ml distilled water and adjust to pH 2 3 2 Make up to 1 0 with distilled water Note Gels should be stored in storing solution at 4 C in a dark location or away from light Deep Purple staining protocols Step Action il Fixing Fix gels from 90 minutes to overnight in fixing solution 2 Staining Stain for 90 minutes at
38. w v Staining 1 Mix 25 ml of silver nitrate solution 2 5 w v with solution distilled water 2 Make up to 250 ml with distilled water Before use Add 0 1 ml formaldehyde 37 w v Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC Solutions 5 Protocols 5 3 Post staining of the gel Preparations Developing solution e Mix 1 packet of sodium carbonate 6 25 g and distilled water e Make up to 250 ml with distilled water Note Stir vigorously to dissolve the sodium carbonate Before use Add 0 2 ml of formaldehyde 37 w v Stop solution 1 Mix EDTA Na3 2H 0 3 65 g with distilled water 2 Make up to 250 ml with distilled water Washing solution Distilled water Preserving solution 1 Mix 75 mlof ethanol and 11 5 ml of glycerol 87 w w 2 Make up to 250 ml with distilled water Silver staining protocol Step Action 1 Fixation Soak the gel in fixing solution for 120 minutes or overnight 2 Sensitizing Discard the solution Add sensitizing solution and gently shake for at least 120 minutes 3 Washing Remove the sensitizing solution Add distilled water and wash five times for 15 minutes each time Leave in washing solution over night 4 Silver reaction Add Staining solution and gently shake for 120 minutes Amersham ECL Gel and Gel Box User Manual 28 9943 33 AC 51 5 Protocols 5 3 Post staining of the gel 52 Step Action Washing
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