Immobiline DryPlate - GE Healthcare Life Sciences
Contents
1. 602 605 P tsch Schneider L and Klein H Carbohydrate analysis of transferrin subfractions isolated by preparative isoelectric focusing in immobilized pH gradients Electrophoresis 4 1992 225 229 G de Jong W L van Noort and H G van Eijk Isoelectric focusing of apolipoproteins on immobilized pH gradients Improved determination of apolipoprotein E phenotypes Electrophoresis 9 1988 576 579 Baumstark M W Berg A Halle M and Keul J Microheterogeneity of apolipoprotein D as revealed by electroblotting following isoelectric focusing in Immobiline DryPlates Electrophoresis 4 1992 262 264 Holmquist L Phenotyping of Apolipoprotein E Immunoblotting After Isoelectric Focussing in Immobilized pH Gradients Electrophoresis 12 1991 59 63 Marz W Cezanne S and Gros W Immobilized pH gradient isoelectric focusing and immunoblotting for investigations of anti Borrelia burgdorferi IgG antibodies Electrophoresis 4 1992 229 234 M Cruz and A Sid n 20 21 22 23 24 Isoelectric focusing in Immobilized pH Gradients applications in Clinical Chemistry and Forensic Analysis review J Chromatogr 569 1991 197 228 Righetti P G Gianazza E Bianchibosisio A et al The application and optimisation of solubilising additives in Immobiline gels Application Note 345 Amersham Biosciences AB Membrane protein analysis by isolectric focusing in immobilized pH gradients Electrophore2. Code No 80 1106 79 to remove all air bubbles from between the glass plate and the support film 5 Mount the gel in the cassette taking particular care that the U frame gasket seals also over the cut off corner of the supporting plastic foil and that the clamps are mounted correctly to avoid leakage 6 Fill the cassette with the desired rehydrating solution 7 Leave the gel to rehydrate for the recommended time 8 Open the cassette and check the gel surface Remove excess liquid by placing a filter paper moistened in distilled water on top of the gel followed by a dry filter paper on top 9 Blot the gel by gently rolling the rubber roller under slight pressure over the dry filter paper Finally remove the filter papers carefully from the gel Since gels rehydrated in detergent containing solutions have less tendency to stick to dry filter paper they can be dried with a simpler procedure Place a piece of dry tissue paper e g Kleenex on the gel press gently to ensure contact between tissue and gel and remove the paper carefully ep9 epld 3 Sample treatment 3 1 Sample preparation Even if Immobiline DryPlate is exceptionally tolerant towards impure samples best results are still obtained with samples that are free from precipitates Should aggregation occur at the application point this can often be overcome by diluting the sample or changing the sample application position Best results are generally obtaine
3. Densitometric evaluation niais teisiin 19 7 References tanesnsted ivedhe ies ates E E 20 8 Ordering information inci 22 ep3 ep4 1 Introduction Immobiline DryPlate offers a convenient and reliable way to obtain the utmost separation power of isoelectric focusing The Immobline system has indefinitely stable pH gradients allowing high voltages for maximal separation and when necessary long focusing times 1 2 The rehydratable dry gels facilitate the use of additives such as urea detergents carrier ampholytes etc for optimal performance even for samples with poor solubility This manual gives general instructions on how to use Immobiline DryPlate for isoelectric focusing Please consult the Application Notes and or the articles in the reference list 3 21 for detailed instructions on specific applications Immobiline DryPlate is a polyacrylamide gel with an immobilized pH gradient It is bound to plastic backing and is ready to use for isoelectric focusing after rehydration The product is available with various pH gradients See Table 1 The pH gradients are linear over the stated interval Table 1 Code No pH Major application Appl Note interval 80 1128 28 4 7 General purpose 80 1128 29 4 2 4 9 a antitrypsin 470 80 1128 30 4 5 5 4 Group specific component 471 80 1128 31 5 0 6 0 Transferrin 472 80 1128 32 5 6 6 6 Phosphoglucomutase 473 ep5 ep6 1 1 Package contents and technical da
4. Triton X 100 Glycerol 87 w w 2 Mercaptoethanol Dithiothreitol DTT e p23 Important Information Immobiline Multiphor MultiTemp PlusOne NovaBlot Sephadex and NAP are trademarks of Amersham Biosciences Limited Amersham and Amersham Biosciences are trademarks of Amersham plc Amersham Biosciences AB 2002 All rights reserved AII goods and services are sold subject to the terms and conditions of sale of the company within the Amersham Biosciences group that supplies them A copy of these terms and conditions is available on request Amersham Biosciences AB Bj rkgatan 30 SE 751 84 Uppsala Sweden Amersham Biosciences UK Limited Amersham Place Little Chalfont Buckinghamshire England HP7 9NA Amersham Biosciences Corporation 800 Centennial Avenue P O Box 1327 Piscataway NJ 08855 USA Amersham Biosciences Europe GmbH Munzinger Strasse 9 D 79021 Freiburg Germany Amersham Biosciences K K Sanken Building 3 25 1 Shinjuku ku Tokyo 169 0073 Japan Amersham e Biosciences Produced by Wikstr ms Sweden 1021704 12 2002 Printed matter Licence 341 051 WY UO
5. epll 1 For 5 20 pl sample volumes Apply sample directly on the gel using Immobiline applicator strip Code No 18 1002 76 This applicator strip makes it possible to use a multiple syringe which allows quick and simple sample loading especially when working with large numbers of samples Check that the contact between the gel and the applicator strip is uniform Leave the applicator strip on the gel during focusing 2 For 15 20 ul and larger sample volumes Use sample application pieces Code No 80 1129 46 Apply the dry pieces to the gel surface at the desired position s in the gradient Using a micropipette apply 15 20 ul volumes of sample solution on each piece To apply larger volumes use 2 or 3 pieces stacked or laid end to end If you want to apply smaller volumes with by this method trim the paper proportionally before applying it to the gel Remove the pieces of paper after completing half the total focusing time 3 For 2 10 ul sample volumes Apply the sample as droplets directly on the gel surface 4 3 Running conditions Place the electrode holder on the Multiphor II electrophoresis unit and align the electrodes with the center of the electrode strips Finally connect the two electrodes to the base unit and place the safety lid in position Connect the electrode leads to the power supply Typical running conditions are listed in Table 3 Table 3 pH range Voltage V Current mA Power W Time KVh Tim
6. film must be removed to allow the current to pass through the gel FilmRemover is ideal for this purpose After the film and the gel have been separated the proteins can be transferred to an immobilizing membrane by using the Multiphor II NovaBlot transfer kit Complete information on running conditions is given in Multiphor II User Manual Code No 18 1103 43 6 Evaluation 6 1 Determination of the isoelectric point Because of the low ionic strength in the gel the pH gradient cannot be directly measured with a standard surface pH electrode unless carrier ampholytes have been included in the rehydrating solution 21 An alternative to direct pH measurement is to run pI calibration proteins in parallel with the experimental samples For details about the use of pI calibration proteins see the Instruction enclosed with each pI Calibration Kit 6 2 Densitometric evaluation ImageMaster 1D Software Code No 80 6350 37 is a powerful software package for protein quantitation and data analysis By using ImageMaster 1D Software together with ImageScanner Code No 18 1134 45 you can capture store evaluate and report all the information contained in your electrophoresis gels ImageMaster 1D Software automatically selects your lanes bands subtracts the background corrects the smiling and integrates areas and band volume OD x mm The software calculates relative amounts percentages of totals and amounts of proteins in real qua
7. the electrode strips by using forceps thereafter immediately immerse the gel in the fixing solution for 60 minutes This solution precipitates the proteins and allow detergents if used to diffuse out Washing Thereafter wash the gel in washing solution for 2 x 15 minutes Incubation Place the gel in incubation solution for a minimum of 40 minutes The gel can be left over night in the incubation solution Washing Thereafter wash three times in distilled water each time for 5 minutes Silver reaction Put the gel in silver solution for 20 minutes Developing Develop the gel in developing solution for 5 15 minutes The protein bands should become intensively dark Stopping Stop the reaction by placing the gel in stop solution for 10 minutes Washing Wash in washing solution for 5 minutes Preserving To preserve the silver stained gel place the gel in preserving solution for 20 minutes Then place the gel on a glass plate with the gelside up and cover the gel with cellophane preserving sheet soaked in preserving solution Allow the gel to dry in room temperature Do not put the gel in a heating cabinett the silver stain will bleach due to the increased temperature 5 2 Coomassie staining This is a general protein stain which detects protein concentrations down to the pg level 250 ml of solution is used in each step Trichloroacetic acid 46 g Fixing solution Sulphosalicylic acid 14 g 30 60 min Make u
8. __ Buctions Immobiline DryPlate User Manual Amersham G 71 7030 01 Edition AD e Biosciences Important user information Reading this entire manual is recommended for full understanding of the use of this product The exclamation mark within an equilateral triangle is intended to alert the user to the presence of important operating and maintenance instructions in the literature accompanying the instrument Should you have any comments on this manual we will be pleased to receive them at Amersham Biosciences AB S 751 82 Uppsala Sweden Amersham Biosciences AB reserves the right to make changes in the specifications without prior notice Warranty and Liability Amersham Biosciences AB guarantees that the product delivered has been thoroughly tested to ensure that it meets its published specifications The warranty included in the conditions of delivery is valid only if the ep2 product has been installed and used according to the instructions supplied by Amersham Biosciences AB Amersham Biosciences AB shall in no event be liable for incidental or consequential damages including without limitation lost profits loss of income loss of business opportunities loss of use and other related exposures however caused arising from the faulty and incorrect use of the product Trademarks Immobiline Multiphor MultiTemp PlusOne NovaBlot Sephadex and NAP are the exclusive trade marks of Am
9. d staining proce dures as well as blotting can also be used 10 12 5 1 Silver Staining This silver staining method is according to Heukeshoven and Dernick 23 with some modifications Silver staining solutions Note 250 ml of solutions are needed per gel All steps should be done with gentle shaking of the tray Fixing solution 60 min Wash 2x15 min Incubation solution minimum 40 min Wash 3x5 min Silver solution 20 min Developing solution 5 15 min Stop solution 10 min Wash 5 min Preserving solution 20 min Trichloroacetic acid Ethanol Dissolve in distilled water and make up to 250 ml Ethanol Acetic acid Make up to 500 ml with distilled water Ethanol Sodiumacetate e 3 H O Glutardialdehyde 25 w v Sodium thiosulfate Na 0 5 H O Make up to 250 ml with distilled water Distilled water Silver nitrate Formaldehyde Make up to 250 ml with distilled water Sodium carbonate Ethanol Formaldehyde Make up to 250 ml with distilled water EDTA Na e 2 H O Ethanol Make up to 250 ml with distilled water Ethanol Make up to 500 ml with distilled water Glycerol 87 w w Ethanol Make up to 250 ml with distilled water Note Add these components immediately before use 30 0 g 125 ml 150 ml 50 ml 75 ml 17 0g 1 3 ml 0 50 g 0 25 g 50 ul 6 25 g 75 ml 25 ul 3 0 g 75 ml 150 ml 25 ml 75 ml epld5 epl6 Fixation Remove
10. d when the samples are solubilized in the rehydration solution If this is not possible the concentration of salt and buffer ions should still be kept at a minimum and as a general rule preferrably below 50 mmol l Excess buffer and salt ions will cause local overheating due to high local currents which can result in protein denaturation and or prolonged running times Desalting and buffer exchange can be carried out by dialysis or more easily by gel filtration using a prepacked Sephadex G 25 column Choose NAP 5 Column NAP 10 Column or PD 10 Column depending on the sample size See Ordering information 3 2 Sample concen tration In general Immobiline gels can take much higher sample loads than corresponding gels with carrier ampholytes Several factors will determine the optimal sample concentration and volume 1 pH range 2 Number and relative proportions of the components in the sample 3 Sensitivity of the detection method used Guide lines PhastGel Blue R stain detects proteins down to the pg range Normally 15 20 pl of sample with a concentration of 0 5 10 mg ml will give good results Silver staining has about 20 times higher sensitivity A suitable load in a narrow pH gradient is normally 2 3 times higher than the load in a pH 4 7 gradient 4 Isoelectric focusing 4 1 Preparing the experiment Setting the cooling temperature Connect Multiphor II electrophoresis unit to MultiTemp II thermostatic circ
11. e h 4 7 3 500 5 0 1 0 15 0 7 15 2 4 4 2 4 9 3 500 5 0 2 0 15 0 15 25 4 7 4 5 5 4 3 500 5 0 2 0 15 0 15 25 4 7 5 0 6 0 3 500 5 0 1 0 15 0 15 25 4 7 5 6 6 6 3 500 5 0 1 0 15 0 15 25 4 7 e p12 Comments Decrease the power and current settings proportionally if only part of the plate is being used The settings above are only to be regarded as guidelines Some proteins focus very slowly and may require as much as 50 60 KVh to give optimal sharp bands This must be determined experimentally in each case Run the sample for different times Since there is no gradient drift in the Immobiline DryPlate there is no limitation in the electrophoresis system as such as to how long the experiment can be continued The limits are set only by the risk of drying out the gel oxidising or denaturing the sample These risks can be minimized by placing a plastic foil on top of the gel running at low tempera tures flushing the unit with inert gas N and or including a reducing agent DTT or b mercapto ethanol in the rehydration mixture The surface can also be protected with DryStrip Cover Fluid 22 epl3 epl4 5 Detection All currently used detection methods can be applied on Immobiline DryPlate gels including Coomassie Blue 4 14 silver staining 23 Possible problems from extensive swelling of the gel can be reduced by adding ethanol 30 to the washing solutions Enzymatic and immunologically base
12. ersham Biosciences AB In view of the risk of trade mark degeneration it is respectfully suggested that authors wishing to use these designations refer to their trade mark status at least once in each article Copyright 1995 Amersham Biosciences AB All rights reserved No part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means without permission in written form from the company Reading this entire manual is recommended for full understanding of the use of this product Contents Li INHFOGUGHIONI assinei 5 1 1 Package contents and technical data 6 2 PreparinetM6 96l aa aa 7 2 1 Preparing the rehydration solution 7 2 2 Opening the package i 8 2 3 Rehydrating Immobiline DryPlate 9 3 Sample treatment i 10 3 1 Sample preparation 10 3 2 Sample concentration cceeeseeeeesteeeeesteeeeesaes 10 4 Isoelectric focusing ccccccccssssccsesssseessnessessesseeessenees 11 4 1 Preparing the experiment 11 4 2 Sample application ii 11 4 3 Running conditions cisien 12 Da DELSCHON picci 14 5il SIVErstainint ctiiia e a 14 5 2 Coomassie Stag seeiis aaa 17 5 3 Electrophoretic transfer i 18 6 EVallalio Nip 19 6 1 Determination of the isoelectric point 19 6 2
13. ntity units using a calibration curve ImageMaster 1D Software also calculates isoelectric points or molecular weights compares bands across different lanes or gels and produces hierarchial clustering information Further lane comparision databasing and identification using an internal library can be done by using ImageaMaster 1D Database Code No 80 6350 94 and an add on package to ImageMaster 1D Software epl9 e p20 7 1 10 IL 12 References Isoelectric focusing in immobilized pH gradients Principle methodology and some applications J Biochem Biophys Meth 6 1981 317 339 Bjellqvist B Ek K Righetti P G et al Righetti P G Immobilized pH gradients Theory and Methodology Vol 20 Laboratory techniques in Biochemistry amp Molecular Biology Elsevier 1990 Analysis of alphal Antitrypsin phenotypes in Immobiline electrofocusing gels Application Note 470 Amersham Biosciences AB Analysis of Gc phenotypes by IEF in Immobiline gels Application Note 471 Amersham Biosciences AB Analysis of the Transferrin phenotypes in Immobiline electrofocusing gels Application Note 472 Amersham Biosciences AB Analysis of Phosphoglucomutase PGM1 phenotypes in Immobiline isoelectric focusing gels Application Note 473 Amersham Biosciences AB Subtyping of group specific component Gc in human semen blood and vaginal fluid by isoelectric focusing in immobilized pH gradients Electrophoresis 9 1988
14. p to 400 ml with distilled water 1 Ethanol 500 ml Destaining solution Make up to 1000 ml with distilled water 5 min 2 Acetic acid 169m Make up to 1000 ml with distilled water Mix 1 1 before use PhastGel Blue R Liebe Coomassie solution Dissolve 1 tablet in 400 ml 10 min destaining solution Stir with a magnetic stirrer and heat the solution to 60 C Filter before use Use only once See above Destaining solution Until the background is clear Glycerol 40 ml Add 360 ml destaining solution and mix well Preserving solution Fixation Remove the elexctrode strips with forceps Immediately place DryPlate in Staining Kit containing fixing solution for 30 60 minutes This solution precipitates the proteins epl7 pl8 Destaining Before staining wash DryPlate in destaining solution for 5 minutes Staining Pour off the destaining solution and stain DryPlate for 10 minutes in staining solution which has been preheated to 60 C Destaining Destain DryPlate with several changes of destaining solution until the background is clear Preserving Place the destained DryPlate in the glycerol preserving solution for 30 60 minutes Then place the gel on a glass plate with the gel side up and cover the gel with a cellophane preserving sheet soaked in preserving solution Allow it to dry at room temperature 5 3 Electro phoretic transfer Before electrophoretic blotting can take place the support
15. radients J Biol Biophys Meth 1986 113 124 Gelfi C Morelli A Rovida E and Righetti P G ep2l 8 Ordering information Designation Code No 80 1128 28 80 1128 29 80 1128 30 80 1128 31 80 1128 32 80 1106 79 18 1004 40 18 1002 76 80 1129 46 17 0851 01 17 0853 01 02 17 0854 01 02 1018 06 1130 05 1102 77 1102 78 1016 86 1013 75 1129 38 80 6350 37 18 1 134 45 17 0518 01 17 0471 01 17 0472 01 17 0473 01 e p22 Immobiline DryPlate pH 4 7 Immobiline DryPlate pH 4 2 4 9 Immobiline DryPlate pH 4 5 5 4 Immobiline DryPlate pH 5 0 6 0 Immobiline DryPlate pH 5 6 6 6 Roller IEF electrode strips 100 Immobiline applicator strip 5 IEF sample application pieces 200 PD 10 column Desalting samples lt 2 5 ml 30 NAP 5 column Desalting samples lt 0 5 ml 20 50 NAP 10 column Desalting samples lt 1 0 ml 20 50 Multiphor Il electrophoresis unit EPS 3500 XL Power Supply MultiTemp Ill Thermostatic Circulator 115 VAC MultiTemp Ill Thermostatic Circulator 220 V AC NovaBlot electrophoretic transfer kit FilmRemover Cellophane preserving sheets 210x320 mm 50 ImageMaster 1D Software ImageScanner PhastGel Blue R Broad pl Calibration kit pH 3 10 Low pl Calibration kit pH 2 5 6 5 High pl Calibration kit pH 5 10 5 Code No Designation PlusOne chemicals 17 1319 01 17 1315 01 17 1325 01 17 1317 01 17 1318 01 Urea
16. sis 6 1985 419 422 Rimpilainen M A and Righetti P G Hybrid isoelectric focusing in rehydrated immobilized pH gradients with added carrier ampholytes Demonstration of human globulins Electrophoresis 6 1985 314 325 Altland K and Rossman U Analysis of recombinant proteins by isoelectric focusing in immobilized pH gradients Electrophoresis 4 1992 214 220 R Bischoff D Roecklin and C Roitsch Apolipoprotein E phenotyping by isoelectric focusing in immobilized pH gradients and silver staining Electrophoresis 4 1992 252 258 R Cartier and A Sassolas Rapid and simple method for the identification of apolipoprotein E isomorphic phenotypes from whole serum Electrophoresis 4 1992 258 262 M KohImeier H J Drossel P Sinha and E K ttgen Swelling kinetics of Immobiline gels for isoelectric focusing Electrophoresis 5 1984 257 262 Gelfi C and Righetti P G Improved rehydration procedure for polyacrylamide gels in presence of urea Demonstration of inhereted presence of prealbumin variants by isoelectric focusing in an immobilized pH gradient Electrophoresis 5 1984 379 381 Altland K Bantzhoff A Hackler R and Rossman U Immobiline DryStrip Kit Instructions Code No 18 1038 63 Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining Electrophoresis 6 1985 103 112 Heukeshoven J and Dernick R pH measurements in ultranarrow immobilized pH g
17. ta Package contents Each gel package contains 3 gels filter papers experimental result forms and instructions Designation Code No No per pack Immobiline DryPlate See label 3 Filter paper 50 Experimental result form 3 Instructions 71 7030 01 1 Technical data Gel dimensions Approx 250x110 x 0 5 mm Gel matrix Polyacrylamide T 4 C 3 Buffering capacity 3 meqv pH L Gel backing Polyester film Storage 20 C Shelf life 18 months from manufacturing Please observe Expiry date printed on each kit Table 2 Alt 1 2 Preparing the gel 2 1 Preparing the rehydra tion solution One of the advantages of the dry gel format is the opportunity to include different additives in the reswelling solution 10 14 16 18 19 The three options given in Table 2 should therefore only be regarded as typical examples that will give good results for most applications However whenever required these recipes can easily be modified for further optimization Consult the relevant Application Note Table 1 and Reference list or any of the cited references for instructions about specific applications The rehydration process itself has also been investigated 20 21 Alt 2 Alt 3 Application areas Composition Distilled water Pharmalyte 3 10 Ampholine pH 3 5 9 5 Urea Triton X 100 DIT Total volume Rehydration time Water soluble Proteins with Proteins with low solubility proteins reduced sol
18. te 2 The gel is packed so that it is faced down to the aluminium foil backing of the package and the gel support is uppermost Note 3 If only half of the gel is to be used cut the package in half with sharp scissors reseal the portion to be saved with tape and store it at 20 C Remember to identify the polarity of the remaining part Open the gel package from the transparent side Use scissors to cut around all four sides of the package taking care not to cut either the gel or its transparent backing film To simplify gel handling later on identify the polarity of the pH gradient The support film has a precut corner which indicates the anodic side of the pH gradient 2 3 Rehydrating Immobiline DryPlate For this procedure the specially designed Reswelling Cassette is highly recommended It allows fast convenient even and reproducible rehydration of the gel It also facilitates including the additives necessary for optimal performance for each application Proceed as follows See the instruction manual for the Reswelling Cassette or the Multiphor II Electrophoresis System user manual for detailed instructions 1 To prevent the gel from adhering to the glass plate fitted with the U frame coat the plate with Repel Silane 2 Mark the cathodic side of the gel 3 Wet a clean thick glass plate with a few drops of water and place the gel on the glass plate with the gel side up 4 Roll the gel with a clean rubber roller
19. ubility e g Membrane proteins Lipoproteins 20 0 ml 19 5 ml 12 0 ml 0 5 ml 0 5 ml 9 6 g 0 1 ml 60 mg 20 0 ml 20 0 ml 20 0 ml 1 2 h 1 2 h 15 18 h Comments Note All chemicals should be of the highest purity PlusOne chemicals are highly recommended Double distilled water should be used The presence of carrier ampholytes not only increases protein solubility but also their electrophoretic migration velocity resulting in shorter focusing times ep7 ep8 Mercaptoethanol 2 or dithiothreitol 15 50 mmol l can be added to avoid oxidation of sensitive proteins Glycerol 20 25 improves solubility of hydrophobic proteins and reduces the risk for urea crystallization Lateral band spreading can be reduced by adding acetic acid 2 mmol l and applying the sample at the anodic side or adding Tris 2 mmol l and applying the sample at the cathodic side Triton X 100 can be replaced with other non ionic or zwitterionic detergents e g CHAPS Other carrier ampholytes than Pharmalyte 3 10 Ampholine pH 3 5 9 5 may also be used Alt 3 in table 2 corresponds to what is used in the first dimension focusing in 2 D electrophoresis This alternative can be regarded as a standard choice for focusing under denaturing conditions and will normally give high quality results with all kind of samples 2 2 Opening the package Note 1 Wear clean gloves to avoid contamination of the gel surface particularly when using silver stain No
20. ulator and set the desired running temperature A running temperature of 10 C is often used except for gels containing urea which are preferably run at somewhat higher temperatures 15 C or more to avoid precipitation of the urea Switch on the thermostatic circulator 15 minutes before starting the run Positioning the gel on the cooling plate Pipette a few milliliters of insulating fluid kerosene or light paraffin oil on the cooling plate of Multiphor II Position the gel on the cooling plate so that the polarity of the gel corresponds with the polarity marked on the cooling plate Ensure that no air bubbles are trapped between the gel and the cooling plate Electrode strips are used to ensure good electrical contact between the gel and the electrodes This prevents sparking and allows salt ions from the gel to migrate into the electode strips where they will stay and not interfere with the separation Soak the electrode strips evenly with approximately 3 ml distilled water and remove the excess by using a filter paper The electrode strips should appear very dry before they are applied to the gel Lay the electrode strips along the long edges of the gel Cut off the parts of the strips which protrude beyond the short ends of the gels using a pair of sharp scissors 4 2 Sample application There are basically three different methods for sample application Which method to choose is determined primarily by the sample volume
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