Hoefer SE 600 Chroma
Contents
1. gel screws facing out in the second cradle in the dual gel caster Attach the gel sandwich to the upper buffer chamber Turn the upper buffer chamber upside down and place a slotted gasket into both sandwich holder recesses Both the slot in the gasket and the slot in the recess must align Both slotted gaskets must be used even if running only one gel sandwich Grooves along each slot help keep the gasket in place Release the sandwiches from the caster by removing all bottom cams if present Lower the upper buffer chamber onto the gel sandwiches in the casting stand Install the cams ridge pointing down into the buffer chamber cam holes Clamp the sandwich in place by simultaneously turning one cam clockwise and the other counterclockwise a fu 180 Note Do not force the cams If you encounter unusual resistance disassemble and inspect clamp and glass alignment along the top of the sandwich Align and reinstall Q Use a pipette to carefully fill each slot above the sample wells with buf fer to minimize disturbing the samples Then pour 100 ml of buffer into the chamber directing the buffer stream toward the side wall Check that no buffer leaks around the gasket Fig 6 Attaching gel sandwiches to the upper buffer chamber If the assembly leaks take it to a sink and partially release the cams to allow buffer to drain out of the upper cham ber Disassemble check alignment of all sandwich component2. 35 then rinse thoroughly with tap and distilled water Glass plates can also be treated with but not stored in acid cleaning solutions Replacing a heat exchanger glass tube Oo Remove the tube by simultaneously twisting and sliding it down as far as possible until the top end is free of the upper grommet Carefully guide the tube so that it will clear the assembly then lift the tube out of the lower grommet Lightly grease the outside of both ends of the new tube with silicone grease Twist and slide one end of the tube into the lower grommet Then slip the other end into the top grommet gently pushing it with a slight twist until it stops 3 Check that the grommet is not pinched 6 p25 problem Gel sandwich leaks while casting possible cause Dirty or damaged components Troubleshooting remedy Plates spacers and the gasket must be completely clean Wash if necessary Replace chipped plates especially if chipped near the spacers Check the caster gasket for cuts or cracks and replace if necessary Mis aligned parts Check plate and spacer alignment realign if necessary Over clamping Turn cam only as far as necessary to create a seal usu ally 90 150 but up to 180 On each spacer apply a light film of Gel Seal compound to the bottom outside corner only Do not use silicone grease Sample Air bubbles Remove air bubbles before inserting combs Slide co
3. manual SE 600 Chroma Hoefer SE 600 Chroma standard dual cooled gel electrophoresis unit um SE600X IM Rev B0 06 08 A tio e f r Page finder Gel electrophoresis unit function and description SP CINCATIONS sscesccescassssssssatsacseaerainsiisaianmaneeataasy 2 Important information 3 Unpacking and Invent 5 Operating instructions Prepare the gel sandwich 9 Construct the gel sandwich amp insert into caster 9 Acrylamide gels 12 Gradient a a Ni 14 Sample preparation and loading 16 Final assembly 18 Separating the sample 22 After electrophoresis 24 Care and maintenance eee 25 Trouble Shooting 26 Bibliography ica an 31 Ordering information sse 34 Companion products 38 pi Gel electrophoresis unit function and description The Hoefer SE 600 Chroma vertical slab gel electrophoresis unit is intended for protein and nucleic acid electrophoresis under commonly used denaturing and non denaturing conditions Up to 28 samples can be compared on a single slab gel Applications include protein separations nucleic acid fractionation and the second dimension separation of 2 D electrophoresis First dimen sion separation of 2 D protein electrophoresis should be performed on Immobilized pH Gra dient Gels The focused strips are easily trans ferred to the second dimension slab gel for size separation The gel plates are 18 cm wide by
4. Check that the gasket is not damaged or pinched Replace if necessary Check that the upper buffer cham ber is not warped from prior exposure to excessive heat Add more silicone grease to seal heat exchanger grommets Check for leaks or cracks in the heat exchanger Replace worn grommets Dye front Uneven heat Fill the lower buffer chamber to the level appropriate for curves up distribution at edges the run See Fig 8 page 21 smiles at edges Use magnetic stirrer and stir bar to Keep buffer well mixed Excessive heat Circulate external coolant Prechill the buffer Decrease the current or voltage setting Run the gel in the cold room p27 problem possible cause remedy Protein Particulates in sample Centrifuge or filter sample before loading to remove par streaks ticulates vertically Overloading Load less sample Degradation Add protease inhibitor such as PMSF Unusually Current leakage around Check for leaks all plates and spacers must be aligned slow gel and free of grease and cracks or fast run If used the buffer dam must be secure Sample or reagent If the required pH of a solution is overshot do not back preparation titrate Discard and prepare fresh buffer Check recipes gel concentrations and buffer dilution For instance do not use Tris HCI instead of Tris for Laemmli tank buffer Decrease the salt concentration of samples Reagent quality Dispose of older acrylamide
5. run Under these conditions voltage increases as the run proceeds A lower current setting is rec ommended for higher resolution The optimal current level must be determined empirically the main factors that must be balanced include the gel concentration and migration speed and the resulting Joule heating and band distor tion Table 2 lists starting point guidelines and adjustments for gel thickness number of gels and migration rate Current Current acts on the total cross section area of all the gels because the gels are connected in parallel in the electrical circuit Thus the cur rent setting for one gel must be multiplied by the number of gels of the same cross section run simultaneously For a gel 1 5 mm thick we sug gest a starting current setting of 25 mA Two 1 5 mm gels 50 mA Note Cooling may be required to control Joule heating Voltage The starting voltage for a 1 5 mm slab gel con nected to a power supply set to 25 mA is usu ally 80 90 V using the SE 600 Chroma unit Caution After initial monitoring do not leave the unit unattended for more than 1 h before checking the progress of the bands and the buffer level with a Laemmli discontinuous buffer system for SDS gels The final voltage is typically 250 400 V depending on the length of the gel See Table 2 Time A run is usually complete when the tracking dye reaches the bottom of the gel In a 16 cm gel SE 600 Chroma a 1 5 mm thick
6. Fill solution to just below the top of the upper plate edge If bubbles are trapped remove with a pipette or syringe Introduce a comb at a slight angle into each sandwich taking care not to trap air bubbles under the teeth Club sandwich Pipette the solution into both sandwiches filling each to the same level below the notched edge Stacking gel Fill solution to 3 4 cm below the top of the glass plate This height allows 1 cm of stacking gel below the wells Pour the gel and apply an overlay see step 2 After the gel is set prepare the stacking gel as described below 2 D electrophoresis Discontinuous protein system Fill monomer solution to about 1 cm below the top of the glass plate to allow 4 5 mm for the IPG strip or tube gel and an agarose seal A stacking gel will require extra space Seal the IPG strip or tube gel in place with agarose dissolved in running buffer Take care to avoid trapping any air bubbles between the first and second dimension gels Overlay each gel with a thin layer of water saturated butanol water or diluted gel buffer to prevent gel exposure to oxygen S owly deliver the overlay solution from a glass syringe fitted with a 22 gauge needle Apply the solution near the spacer at one side of the sandwich and allow it to flow across the surface unaided Allow the gel to polymerize for a minimum of 1 h Stacking gel preparation Pour the stacking gel while the sandwich is still in
7. Proteins of human urine I Concentration and analysis by two dimensional electrophoresis Clin Chem Jul 25 7 1199 2210 1979 Anderson Leigh and Anderson Norman G High resolu tion two dimensional electrophoresis of human plasma proteins Proc Natl Acad Sci USA 74 5421 5425 1977 Anderson L Two Dimensional Electrophoresis Operation of the ISO DALT System Second Edition Large Scale Biol ogy Press 1991 Bravo R Schafer R Willecke K MacDonald Bravo H Fey S J and Celis J E More than one third of the discernible mouse polypeptides are not expressed in a Chinese hamster mouse embryo fibroblast hybrid that retains all mouse chromosomes Proc Natl Acad Sci USA Apr 79 7 2281 2285 1982 Hurkman W J and Tanaka C K Solubilization of Plant Membrane Proteins for Analysis by Two Dimensional Gel Electrophoresis Plant Physiology 81 802 806 1986 Mets L J and Bogorad L Two dimensional polyacrylamide gel electrophoresis an improved method for ribosomal proteins Anal Biochem Jan 57 1 200 210 1974 O Farrell P H High resolution two dimensional electropho resis of proteins J Biol Chem May 25 250 10 4007 4021 1975 6 p32 Bjellqvist B et al Isoelectric focusing in immobilized pH gradients principle methodology and some applications J Biochem Biophys Methods 6 317 339 1982 Gorg A et al The current state of two dimensional electro phoresis wi
8. as necessary for variables such as ambient temperature changes in power output and circulator bath efficiency If accurate temperature control is critical measure the temperature and adjust as necessary Optional Prechill the buffer Fit the upper buffer chamber assembly into the lower buffer cham ber Use a steady hand to avoid disturbing the samples Grasp the assembly in the casting stand by the upper buffer chamber and carefully lower it into the lower chamber Q Inspect the installation and check the buffer levels Upper buffer chamber UBC The electrode along the upper chamber ridge must be submerged about 1 cm This level requires 450 600 ml of buffer just enough to cover the upper chamber ribs but not high enough to contact the banana plug Do not fill above UBC MAX fill line Lower buffer chamber LBC Fill to LBC MAX fill line 6 p19 Important assembly notes e JEF runs The buffer level in the lower buffer chamber must never reach the upper buffer chamber maintain at least 2 cm of clearance e Do not fill the upper or lower chamber above the recommended levels illustrated on the next page Remove buffer in contact with the electrode posts e Pour buffer slowly and away from the slots in the upper buffer cham ber to avoid disturbing the samples e Use only water or 50 50 water eth ylene glycol as coolant Never use a commercial antifreeze or any alcohol based mixture or irrepa
9. exchanger to a water tap or any coolant source where the water pressure is unregulated Do not operate with buffer temperature above 45 C All plastic parts are rated for 45 C continuous duty Circulate coolant through the heat exchanger during electrophoresis to minimize heating Overheating will cause irreparable damage to the unit e Only accessories and parts approved or supplied by Hoefer Inc may be used for operating maintaining and servicing this product Informations importantes e Le couvercle de s curit doit tre en place avant de brancher les prises au g n rateur e Eteindre le g n rateur et d brancher les prises avant d enlever le couvercle de s curit e Faire circuler seulement de l eau ou 50 50 d eau et d thyl ne glycol dans l changeur vertical cirulation d eau Ne jamais utiliser d anti gel ou tout autre solvant organique avec cet instrument Les solvants organiques causeraient des dommages irr parables l appareil e Ne pas connecter l changeur vertical circulation d eau un robinet ou quelque source de refroidissement dont la pression n est pas r guli re e Ne pas utiliser avec un tampon une temp rature au dessus de 45 C Toutes les pi ces en plastique sont pr vues pour r sister une temp rature constante de 45 C Faire circuler l eau dans l changeur vertical durant l lectrophor se pour minimiser l chauffement afin d viter des dommages irr
10. rable damage to the heat exchanger will result e Do not connect the heat exchanger to a water tap or any other source where the water pressure is unregu lated Fig 7 Approximate circulator bath temperature setting Set the circulator bath temperature setting lower than the desired run temperature by the amount indi cated on the graph This should be checked at three points p20 8 g 2 3 BD S E a E 0 10 20 30 40 50 60 power supply setting W Example Run parameters 200 V 0 05 A 50 mA 1 Calculate W if your power supply does not display power directly W VxA 10 W 200 V x 0 05 A 2 Interpolate the number of degrees to subtract from the desired run temperature 10 W intersects the graph at about 1 C If the desired temperature is 23 C set the bath to 23 1 22 C If the desired temperature is 4 C set the bath to 4 1 3 C Fig 8 Upper and lower buffer chamber fill levels Upper chamber usc buffer max fill line MAX Lower chamber buffer max fill line Buffer level iSe label MAX Place the safety lid on the unit by engaging the safety interlock pins before lowering the electrode connections on to the banana plugs 6 Plug the color coded leads into the jacks of an approved power supply Plug the red lead into the red output ja
11. solutions and use only stock of the highest quality Use only freshly deionized urea Voltage or current To increase or decrease the migration rate adjust the settings voltage or current by 25 50 Bands are Incomplete gel prepa Degas the stacking gel solution and avoid trapping air skewed or ration and polymeriza bubbles under the comb teeth distorted tion Irregular interface Overlay the running gel with water saturated butanol between stacking and before polymerization begins to avoid forming an uneven running gels gel surface Sample preparation Dialyze or desalt the sample 6 p28 problem possible cause remedy Stained sample collects Near the Gel concentration Molecules are not sufficiently restricted by the resolving buffer front gel pore size increase the T Degradation Proteins may be degraded by endogenous proteases use protease inhibitors during the isolation step Near the top Gel concentration The gel pore size is too small decrease the T of the of the gel i i aher ths resolving or stacking gel buffer front ae has reached Precipitation The protein has precipitated Heat the sample at a lower the bottom temperature 70 or less for 1 2 min At both top Gel concentration The molecular weight range of the sample requires an and bottom acrylamide concentration gradient to resolve the full range of the gel Le of protein sizes Poor band Running Begin electrophoresis as soon as the sample is loaded to resol
12. the gel caster Stacking gel resolution is optimal when poured just before elec trophoresis Q Remove the overlay by rinsing the top of the gel several times with distilled water Invert the caster to drain To ensure a seamless contact between the resolving and stacking gels remove residual liquid by blotting one corner with a lab wipe 5 Calculate the stacking gel monomer solution volume O Prepare the stacking gel monomer solution deaerate it and add catalyst and initiator Pour the stacking gel onto the resolving gel with a disposable or Pasteur pipette to a level about 2 mm from the top of the plate Q Introduce a comb at a slight angle into the sandwich taking care not to trap air under the teeth Allow a minimum of 1 h for the gel to polymerize e p13 Fig 5 Pouring a gradient gel A pipette tip may be used instead of a cannula if the gel solution is delivered at a rate that maintains a continuous stream on the glass surface Note Gradient gels poured in the SE 615 or SE 675 Multiple Gel Caster are introduced through the bottom Note When pouring an exponential gradient gel position a plunger or sealing plug above the liquid in the mixing chamber to hold the volume constant p14 Gradient gels Both linear and exponential gradient gels can be poured in the dual gel caster We recommend using a Hoefer SG Series Gradient Maker Gradient gels are poured from the top of the caste
13. 03U Clamp assemblies 16 cm SE6003U Clamp assemblies 8 cm SE6403U Glass plates 18x 8 cm Glass plates 2 SE6402 Glass plate club sandwich divider notched 1 SE6402D 18 x 16 cm Glass plates 2 SE6102 6 p36 product quantity product number Glass plate club sandwich divider notched I SE6102D Teflon combs number thickness width of wells mm mm 10 0 75 8 3 SE511 10 75 10 1 00 8 3 SE511 10 1 0 10 1 50 8 3 SE511 10 1 5 15 0 75 5 7 SE511 15 75 15 1 00 5 7 SE511 15 1 0 15 1 50 5 7 SE511 15 1 5 20 0 75 4 1 SE511 20 75 20 1 00 4 1 SE511 20 1 0 20 1 50 4 1 SE511 20 1 5 28 0 75 2 7 SE511 28 75 23 1 00 2 7 SE511 28 1 0 23 1 50 27 SE511 28 1 5 Comb depth 15 mm all others 25 mm Preparative combs These combs are 25 mm deep adjustable to 10 or 15 mm no of wells thickness width mm prep ref mm prep ref 1 1 0 75 121 6 SE511 R 75 1 1 1 121 6 SE511 R 1 0 1 1 1 50 121 6 SE511 R 1 5 1 2 0 75 113 6 1 SE511 DR 75 1 2 1 113 6 1 SE511 DR 1 0 1 2 1 50 113 6 1 SE511 DR 1 5 Adjustable comb back 1 SE511 BKA Required to convert any 25 mm deep comb to 10 or 15 mm depth p37 Spacers thickness length width qty product number mm cm cm 0 75 8 2 2 SE6419 2 75 0 8 2 2 SE6419 2 1 0 5 8 2 2 SE6419 2 1 5 0 75 16 2 2 SE6119 2 75 0 16 2 2 SE6119 2 1 0 5 16 2 2 SE6119 2 1 5 0 16 1 2 SE6118 2 1 0 5 16 1 2
14. 16 cm long Up to four gels can be run at one time if sand wiches are paired into club sandwiches The heat exchanger allows buffer temperature con trol in the lower chamber Specifications Gel plate size 18 x 16 cm w x h Gel size 14 or 16 cm x 16 cm w x h Maximum watt 50 W Maximum volt 1 000 V Maximum ampere 500 mA Maximum temperature 45 C Environmental operating conditions Indoor use 4 40 C Humidity up to 80 Altitude up to 2000 m Installation category II Pollution degree 2 Dimensions width x height x depth 32 x 29 x 14cm 12 5 x 11 5 x 5 5 in Product certifications EN 61010 1 UL 61010A 1 CSA C22 2 1010 1 CE Cer tified This declaration of conformity is valid only when the instrument is e used in laboratory locations e used as delivered from Hoefer Inc except for alterations described in the user manual and e connected to other CE labeled instruments or products recom mended or approved by Hoefer Inc gt gt Important information e The safety lid must be in place before connecting the power leads to a power supply e Turn all power supply controls off and disconnect the power leads before removing the safety lid e Circulate only water or 50 50 water ethylene glycol through the heat exchanger Never introduce antifreeze or any organic solvent into any part of the instrument Organic solvents will cause irreparable damage to the unit Do not connect the heat
15. Laemmli SDS gel run at 25 mA gel without cooling usually requires 5 h Record each run Keep a record of the current or voltage setting number and thickness of gels buffer system and the starting and final current or voltage readings for each run so that results can be compared Inconsistent results for the same system and settings indicate potential problems such as leaking current incorrect buffer concen trations high salt concentrations or inconsis tent chemical quality Check band progress after 5 min and again after 1 h keeping an eye on the migration rate of the tracking dye The run is complete when the tracking dye reaches the bottom of the gel Watch the buffer level and if necessary replen ish it as required to keep the top electrode submerged A small volume of buffer may leak past a nicked plate or gasket or buffer may pass through the gel 6 p23 6 p24 After electrophoresis Once the tracking dye reaches the bottom of the gel turn off the power supply disconnect the leads and remove the safety lid using finger leverage between the lid and the top of the heat exchanger Lift straight up to avoid bending the banana plugs 2 If coolant is circulating stop the flow and disconnect the fittings or tubing Pull out the upper buffer chamber assembly Pour the buffer into a sink Install the assembly in the dual gel caster and then release the sandwiches by turning and removing
16. SE6118 2 1 5 Companion products Hoefer SE 100 Plate Mate washing and storage unit 1 SE100 QuickFit connectors female 3 8 2 QF3 8 QuickFit connectors male 3 8 2 QFX3 8 6 p38 Hoefer Inc 953 Indiana Street San Francisco CA 94107 USA www hoeferinc com Hoefer is a registered trademark of Hoefer Inc Coomassie is a trademark of ICI plc Teflon is a trademark of E l du Pont de Nemours amp Co 2008 Hoefer Inc All rights reserved Printed in the USA Hoefer
17. V Banana plug gold with 2 washers 1 SE6067 SE 600 Chroma Heat exchanger lower electrode assembly 1 SE6160 Glass tube with 2 grommets for heat exchanger lower electrode assembly 1 SE6160 5 Grommets for heat exchanger lower electrode assembly 4 SE6060 6 aan vad Spirit level 1 SER11 SE6150X Gel Seal compound 1 4 oz tube 1 SE6070 Spacer Mate 3 SE6119SM p34 product qty product number Gel casters For 1 or 2 gels Dual Gel Caster basic 2 gels 18 cm wide 1 SE6015 Includes 2 blank gaskets for 1 or 2 gels One included with each SE 600 Chroma unit For up to 4 gels Gel Caster Kit 4 gels 18 x 16 cm 1 SE675 Includes 8 glass plates 3 space saver plates 5 filler sheets 100 sheets of wax paper Spacer Mate alignment template and filler plugs Order combs and spacers separately For up to 10 gels Multiple Gel Caster Kit 10 gels 18 x 16 cm 1 SE615 Includes 20 glass plates space saver plate 5 filler sheets 100 sheets of wax paper Spacer Mate alignment template and filler plugs Order combs and spacers separately 6 p35 spirit level choose the appropriate spacer and plate length for your unit universal clamp SE6003U SER11 L cam SE6005L Clamps and cams Clamp and Cam Kit four 16 cm clamps and 8 black cams 1 SE6003UK Replacement thumbscrews for clamps 12 SE6003U 2 Cams black for clamps with cam holes 64
18. ated by the spacers and plates if the sandwiches tend to leak O Place the laminated gasket into the casting cradle See Fig 4 with the foam side down Place the clamp assembly in the casting cradle screw side facing out U Insert a cam into the hole on each side of the casting tray with the ridge short end pointing up Seal the gel sandwich against the casting gasket by turning both cams as far as needed usually 90 150 up to 180 The cam action presses the plates down into the gasket to seal the bottom of the sandwich The seal is complete Note When turning the cams it is easier to keep the caster balanced if you turn both toward the center of the caster Fig 4 Caster components and setup gasket foam side down glass plate once the glass edge appears darker and nearly transparent against the gasket Do not turn past this point spacer cam hole clamp cam hole casting cradles 2 cam hole leveling feet 4 cam install ridge end up cam hole epll 6 p12 Acrylamide gels Prepare the monomer solution and pour the gel Prepare the required amount of monomer solution Deaerate and add the initiator and catalyst just prior to pouring the gel Pipette the solution into one corner of the sandwich taking care not to intro duce any air bubbles See below for the appropriate solution level according to the application No stacking gel Continuous system
19. ck and the black lead into the black output jack In most systems the red lead which is connected to the bottom electrode is the anode and the black lead connected to the top electrode is the cathode p21 Note SE 600 Chroma unit uses 18 cm wide plates The gel thickness determines the cross section and current requirement for constant current runs The length of the plate determines the running time Table 2 Laemmli buffer system Starting point guidelines Gel thickness 1 5 mm Current per gel 25 mA con stant current Starting voltage 80 90 V 220 250 V Final voltage Thicker or thinner gels require proportionally more or less current For example a 0 75 mm gel which is half as thick as a 1 5 mm gel requires half as much current or 12 5 mA The current must be multiplied by the number of gels For instance if two club sandwiches are installed the four gels require four times as much current The current can be increased for faster runs if active cooling is used and it can be decreased for slower over night runs At 25 mA per gel 6 p22 Separating the sample Electrophoresis parameters for discontinuous polyacrylamide gels Gels may be run at either constant current or constant voltage settings A constant current mode is traditionally used with a discontinuous buffer system so that the rate of electrophoretic migration remains unchanged throughout the
20. does not introduce bubbles into the solution Mix the gradient and pump the solution into the sandwich While the solution is stirring begin pumping from the mixing chamber and open the stopcock to the reservoir chamber Raise the cannula as liquid enters the sandwich keeping the tip at the gel surface Prepare more gels as required O Overlay each gel with a thin layer of water saturated butanol water or diluted gel buffer to prevent gel exposure to oxygen S owly deliver the overlay solution from a glass syringe fitted with a 22 gauge needle Apply the solution near the spacer at one side of the sandwich and allow it to flow across the surface unaided Q Allow the gels to polymerize for a minimum of 1 h After polymer ization pour off the overlay and rinse the gel surface several times with distilled water 8 Prepare the stacking gel monomer solution pour the stacking gel and introduce a comb at a slight angle into the sandwich taking care not to trap air under the teeth Allow a minimum of 1 h for the gel to polymerize pl5 Note With Coomassie Blue it is possible to detect 1 ug of protein in a single band With the more sensitive silver stains it is possible to detect as little as 10 ng of protein Note Once the samples are in the wells take care to not jar the sand wiches so that the samples are not spilled or mixed 6 pl6 Sample preparation and loading The sample can be
21. es and two spacers Lay one plate on a flat surface lay the Spacer Mate alignment template onto the plate wide side at the top of the plate place a spacer along each edge and lay the second glass plate on top Secure the sandwich with clamps Slide one clamp at a time along the sandwich sides Finger tighten one screw on each clamp set the sandwich upright on a flat surface and loosen the screw to align the stack Taking great care in alignment will ensure a good seal Finger tighten all screws Remove the Spacer Mate Tip Use the casting cradle to hold the sandwich during align ment Remove the laminated gasket from the cradle and instead of setting the sandwich upright on a flat surface set it into the casting cradle Q Club sandwich 16 cm long notched center divider plate ordered separately pairs two sandwiches to double the number of gels that can be cast and run Assemble a club sandwich in the same manner as a regular sand wich except before placing the top glass plate lay the divider plate and a second set of spacers on the stack Place the notch so that it will be at the top of the gels It is essential that the spacers and plates align perfectly in order to seal 5 Remove the sandwich and inspect the bottom to make sure that edges are aligned flush to ensure a complete seal Adjust if necessary Optional Apply a light film of Gel Seal compound only on the bottom corner surfaces cre
22. es the height of the sample sharpen into in the well a concen trated zone Reagent quality in the stack Dispose of outdated acrylamide solutions and use only the highest grade of acrylamide ing gel Sample preparation 6 p30 When preparing samples avoid using solutions with high salt concentrations Bibliography General Gallagher S R and Smith J A Electrophoretic separation of proteins In Current Protocols in Molecular Biology Ausubel F A eds OSC 10 2 1 10 2 21 1991 Hames B D and Rickwood D Gel Electrophoresis of Proteins A Practical Approach Second edition City IRL Press 1990 Sambrook J and Russell D W Molecular Cloning A Labo ratory Manual Cold Spring Harbor Laboratory Cold Spring Harbor NY 2001 Sasse J and Gallagher S R Staining proteins in gels Current Protocols in Molecular Biology Ausubel F A et al eds OSC 10 6 1 10 6 8 1991 SDS Polyacrylamide Gel Electrophoresis and Isoelectric Focus ing Handbook 80 6013 88 Hoefer Inc 2001 Non denaturing gel systems Reisfeld R A et al Acidic buffer system for resolution of cationic proteins Nature 195 281 1962 McLellan T Electrophoresis buffers for polyacrylamide gels at various pH values Anal Biochem 126 94 1982 Hedrick J L and Smith A J Size and charge isomer separa tion and estimation of molecular weights of proteins by discontinuous gel electrophoresi
23. esis process The chamber is chemically resistant to common electrophoretic buffers but not to organic solvents or strong acids or alkali Tem peratures above 45 C may cause the chamber to warp Upper buffer chamber The upper buffer chamber is chemically resistant to common electrophoresis buffers but not to organic solvents or strong acids or alkali The upper electrode cathode runs along the center ridge and terminates at the banana plug The upper chamber requires 0 5 0 8 of buffer fill no higher than the top of the plastic ribs Heat exchanger The heat exchanger must be installed for every use because it houses the bottom electrode anode which runs along the bottom of the frame When connected to a circulator bath the heat exchanger regulates the buffer temperature in the lower chamber Coolant passes through the glass tubes which are secured with silicone rubber grommets The heat exchanger connec tor ports are 13 mm o d The heat exchanger is rated to a maximum of 0 8 atmospheres above ambient 12 psig Connect only to coolant sources with regulated pressure Do not con nect to the water tap Safety lid The banana plug on the heat exchanger connects to the red lead and the plug on the upper buffer chamber connects into the black lead The 4 mm shrouded color coded leads plug into color coded jacks in the power supply Engage interlock pins before lowering electrode connections on to banana plugs Alwa
24. g water for 90 s then allow to cool to room temperature Treated samples can be stored at 40 to 80 C for future runs Heat membrane proteins to 60 C for 20 min Store unused sample at 4 C Q Underlay the sample into the wells using a fine tipped microsy ringe or gel loading pipette tip Table 1 Sample volume for standard comb sizes volume of sample ul per 1 mm depth no of comb thickness mm wells 0 75 1 0 1 5 10 6 2 8 3 12 4 15 4 3 5 7 8 6 20 3 1 4 1 6 2 28 2 1 21 4 1 1 1 ref prep 4 90 6 121 9 183 1 2 ref prep 4 85 6 112 9 171 p17 Note To help hold the gasket against the upper buffer chamber dab a small amount of Gel Seal at each end of the gasket on y and then install Important smooth fit between the sandwich and gasket is essential to a good seal 6 p18 Final assembly Upper buffer chamber Rinse both buffer chambers with water and distilled water thoroughly before each use Note Before using the first time disassemble the unit and wash with a dilute solution of a labora tory detergent and rinse thoroughly first with water and then with distilled water Clean away any gel adhering to the exterior of the gel sandwiches 2 If running only one gel Block the second upper buffer cham ber slot by installing the acrylic buffer dam included with the unit Fit clamps onto the dam taking care to align the clamp ends and dam edges Install the dummy
25. his template aligns spacers during sandwich assembly Combs Teflon combs are available in sizes that form 10 15 20 or 28 wells Most combs are avail able in all three thicknesses 0 75 1 and 1 5 mm Blank combs form a single large well and preparative combs include one or two reference wells in addition to the preparative well May be ordered separately All blanks preparative combs and 10 15 and 20 well combs form wells that are 25 mm deep The 28 well comb forms wells that are only 15 mm deep so that wells do not collapse when the comb is removed The sample volume held by each well depends on the gel thickness well depth and the number of wells per comb Table 1 lists sample volumes of wells for all combs see page 17 Wonder Wedge Gel Plate Separation tool This tool is used to disassemble gel sandwiches and to check spacer and comb thicknesses Operating instructions Gel casting and electrophoresis procedures follow Included are instructions for polyacryl amide gels used with continuous or discon tinuous buffer systems and gradient gels See page 31 for bibliography Prepare the gel sandwich Glass plates spacers and clamp sets are sized so that the assembled sandwich can be easily aligned to create the seal required first to cast the gel and then to run it For best results take extra care to align all components when assembling sandwiches One to four gels 18 x 16 cm can be assembled and ru
26. loaded either while the sandwich is in the caster or after the upper buffer chamber is attached When loading samples while using divider plates the samples must be loaded Without the upper buffer chamber in place The amount of sample loaded depends on the thickness of the gel the sensitivity of the detection method used and the amount of sample expected in each band In a continuous buffer system the protein sample should be relatively concentrated because no stack ing gel is used In a discontinuous buffer system the zone into which each molecular species migrates is sharpened by the stack ing gel so the sample need not be as concentrated Prepare the wells Remove the comb by gently rocking it side to side and then lifting it straight up to avoid damaging the well walls Carefully rinse each well with distilled water to remove unpolymerized acrylamide and then drain by inverting the gel sandwich or caster Fill each well with electrophoresis buffer 2 Prepare the sample Increase liquid sample density with 10 glycerol or sucrose Add a tracking dye such as phenol red bromophenol blue or pyronin Y For SDS protein gels use 2X treatment buffer to denature both liquid and dry samples in a test tube To liquid protein solutions add an equal volume of 2X buffer To dry protein samples add equal volumes of 2X sample buf fer and high purity water to achieve the desired concentration 3 Heat the tube in boilin
27. mb wells into solution at an angle If comb must be removed add damaged or more monomer solution before reinserting the comb irregular Incomplete or delayed Allow acrylamide gels to set for a minimum of 1 h polymerization Debris in wells Rinse out unpolymerized gel with sample buffer Comb removal Remove the comb at a slight angle and very slowly to pre vent damaging the gel Agarose gels Lower the comb no more than 1 cm into the gel Incomplete Chemicals Use only recent stocks of the highest quality reagents gel polymer ization If the dry ammonium persulfate does not crackle when added to water replace with fresh stock Increase TEMED or APS concentration or both SS p26 problem Upper buffer chamber leaks Power supply detects current leak possible cause pH remedy Solutions with extreme pH values especially acidic may not polymerize Oxygen Remove oxygen from the gel environment Degas the monomer solution 5 10 min before pouring and then overlay the gel surface with water saturated n butanol Temperature Adjust the gel solution temperature to a minimum of Mis aligned parts 20 C especially for low T gels Check that the glass plates spacers and clamps are aligned and fit snugly into the upper chamber gasket Check that both gaskets are centered and that the posi tioning ridges fit inside the grooves Dirty or damaged components Electrical path to outside ground earth
28. n in the SE 600 Chroma Both precast gels and self cast gels can be used To self cast multiple gels kits can be ordered separately The SE 615 Multiple Gel Caster Kit holds up to 10 single gel sandwiches and the SE 675 Gel Caster Kit holds up to four sandwiches See the accompanying gel caster User Manual for complete instructions To run four gels con currently two accessory notched divider plates and two additional pairs of spacers are required Construct the gel sandwich and insert into caster Prepare the caster and clamps Place the spirit level into the caster center and adjust the leveling feet Loosen all clamp screws and make space for the sandwich by sliding the pressure plates toward the screws Fig 2 Sandwich assembly Inspect glass plates for nicks Use only unchipped plates to prevent leaking spacer glass plates clamp ridges pressure plate O The glass plates and spacers must be flush with the clamp ridges at both top and bottom for a good seal Note Do not use silicone grease or petroleum jelly to seal the sandwich These substances are difficult to remove and ultimately cause artifacts Fig 3 Club sandwich assembly Side clamps will accommodate two spacers up to 1 5 mm thick glass plates at the outer sides of the sandwich spacers ma notched center plate 6 plo 2 Construct gel sandwiches For each sandwich choose two perfectly clean unchipped glass plat
29. parables l instrument e Seulement les accessoires et pi ces detach es approuv s ou fournis par Hoefer Inc sont recommand s pour l utilisation l entretien et r paration de cet appareil Fig 1 Main components of the color coded Hoefer SE 600 Chroma aada 9 see Fig 4 for caster compo nents safety lid Included but not shown e Gel Seal compound 1 4 oz e Spacer Mate alignment template Glass plates 6 e Wonder Wedge plate separation tool interlock pins e Buffer dam Complete unit also includes upper spacers 4 and combs 2 buffer chamber Required but not included with upper e Magnetic stirrer electrode e Power supply with a minimum rating of 500 V 100 mA constant A or V Optional Circulator bath heat exchanger Note The ordering section lists with lower all accessories and replacement electrode parts lower buffer chamber Unpacking and inventory Unwrap all packages carefully and compare contents with the packing list making sure all items arrived If any part is missing contact your local sales office Inspect all components for damage that may have occurred while the unit was in transit If any part appears dam aged contact the carrier immediately Be sure to keep all packing material for damage claims or to use should it become necessary to return the unit Lower buffer chamber The lower buffer chamber is transparent which allows visual tracking of electrophor
30. r with a cannula if using the provided dual gel caster or from the bottom if using a Hoefer Multiple Gel Caster see instructions accompanying the caster A stacking gel is then poured over the gradient gel Pouring a linear gradient gel B o Q o o Assemble sandwich es into the dual gel casters as described in section 3 11 2 Set up the monomer solution flow path Run a length of clear vinyl tubing through a peristaltic pump Attach one end of the tubing to the gradient maker outlet port and the other end to a 20 cm cannula The o d of the cannula must be less than the spacer thickness Place the cannula so that it rests at the bottom of the sandwich midway between the spacers 3 Prepare the monomer solution Calculate the volume of monomer solution needed Divide the total volume in half and prepare this volume of both the higher and lower percentage acrylamide solutions Optional Adjust the higher percentage acrylamide solution to 15 w v sucrose or 25 v v glycerol to improve layering _ O Pour the light solution into the reservoir chamber the chamber farthest from the outlet Open the stopcock between the chambers long enough to displace the air and then close Pour the heavy solution into the mixing chamber and place a stirring bar into this chamber Place the gradient maker onto a magnetic stirrer and begin stirring at a rate that mixes well but
31. s Arch Biochem Biophys 126 155 1968 Denaturing gel systems Laemmli U K Cleavage of structural proteins during the assembly of the head of bacteriophage T Nature 227 680 685 1970 Matsudaira P T and Burgess D R SDS microslab linear gradient polyacrylamide gel electrophoresis Anal Biochem 87 386 396 1978 Schreier M H Erni B and Staehelin T Initiation of mammalian protein synthesis I Purification and charac terization of seven initiation factors J Mol Biol Nov 116 4 727 753 1977 6 p31 EE Shapiro A L and Maizel J V Jr Molecular weight estima tion of polypeptides by SDS polyacrylamide gel electro phoresis further data concerning resolving power and general considerations Anal Biochem Jun 29 3 505 514 1969 Schaegger H and Von Jagow G Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa Anal Biochem 166 368 379 1987 Weber K and Osborn M The reliability of molecular weight determinators by dodecyl sulfate polyacrylamide gel elec trophoresis J Biol Chem 224 4406 4412 1969 Two dimensional electrophoresis Adams L D and Gallagher S R Two Dimensional Gel Electrophoresis Using the O Farrell System Current Protocols in Molecular Biology Ausubel F A et al eds OSC pp 10 4 1 10 4 13 1992 Anderson N G Anderson N L and Tollaksen S L
32. s and adjust if necessary A Remove cams from the lower cam holes Place the upper cham ber onto the sandwiches and then insert the cams into the upper cam holes ridge short end pointing down B The final cam position not shown must be vertical so that the assembly fits into the lower buffer chamber Lower buffer chamber U Place a magnetic spin bar into the lower buffer chamber LBC and place the unit on a magnetic stirrer Fill the lower chamber with up to 4 liters of buffer 2 Lower the heat exchanger into the lower chamber fitting the ports into the notches in the rim The heat exchanger must be in place for all runs because the lower electrode is integrated into the heat exchanger If no cooling is required skip to step 3 Optional Connect the heat exchanger to a thermostatic circulator Slide hose clamps four total onto each end of two lengths of 10 12 mm i d 3 8 1 2 vinyl or silicone tubing Attach one end of each length of tubing to a heat exchanger port Attach the free ends of each length of tubing to the circulator bath ports one to the inlet and the other to the outlet Secure the connections with the hose clamps Note f the cooling option is used frequently it is convenient to attach QuickFit connectors to the tubing The valves in these fit tings prevent coolant spillage Use the chart below to estimate a starting point for the circulator bath temperature setting Adjust
33. th immobilized pH gradients Electrophoresis 9 531 546 1988 Gorg A Two dimensional electrophoresis with immobilized pH gradients current state Biochem Soc Trans 21 130 132 1993 Bjellqvist B et al Micropreparative two dimensional elec trophoresis allowing the separation of samples contain ing milligram amounts of proteins Electrophoresis 14 1375 1378 1993 Blomberg A et al Interlaboratory reproducibility of yeast protein patterns analyzed by immobilized pH gradient two dimensional gel electrophoresis Electrophoresis 16 1935 1945 1995 6 p33 ww Ordering information product qty product number SE 600 Chroma complete unit 1 SE600X 15 1 5 safety lid with cables SE6056X Includes 3 sets of glass plates two 15 well combs 2 sets of spacers 1 5 mm thick 6 cams dual gel casting stand with leveling base and level buffer dam Spacer Mate alignment template and Wonder Wedge Gel Plate Separation tool Replacement Parts Wonder Wedge Gel Plate Separation tool 1 SE1514 bee Slotted silicone rubber gaskets chamb r for upper buffer chamber 2 SE6008B SE6054 Laminated silicone rubber gaskets for casting stand 2 SE6009 Buffer dam 1 SE6032 Upper buffer chamber for SE 600 Chroma 1 SE6054 heat exchanger Lower buffer chamber for SE 600 Chroma 1 SE6150X SE6160 Lid with high voltage leads for SE 600 Chroma 1 SE6056X High voltage safety lead set 1 SE6056 H
34. the cams 4 Unscrew the clamps from the sandwiches and remove Gently loosen and then slide away both spacers Use the Hoefer Wonder Wedge Gel Plate Separation tool to separate the plates Carefully lift the glass plate with the gel attached Handle the gel with care to avoid damaging it Invert the plate and position the gel low over the staining tray Pry one corner of the gel away from the glass and allow it to drop into the tray or if the gel is thick enough to handle lift it and place it into the tray To avoid splashing add staining or fixative solution to the tray after the gel is transferred G Clean the unit as described in the next section Cleaning e Do not autoclave or heat any part above 45 C e Do not use organic solvents abrasives strong cleaning solutions or strong acids or bases to clean the chambers e Do not soak the laminated gasket Note If the old tube is cracked or broken protect your hand with thick gloves a piece of cloth or paper tow els before removing the tube Care and maintenance Immediately after each use rinse the upper and lower buffer chambers with water and then rinse thoroughly with distilled water Handle the upper buffer chamber with care to prevent damaging the banana plug Clean gaskets with mild detergent and rinse with distilled water Allow to air dry Clean glass plates and spacers with a dilute solution of a laboratory cleanser such as RBS
35. ution conditions prevent low molecular weight species from diffusing Conduct the separation at a lower current or voltage set ting to reduce Joule heating Reagent quality Use only the highest quality reagents Poor stacking Use only gels that were recently prepared Add a stacking gel or increase height of the stacking gel Prepare the resolving gel surface by first rinsing it with stacking gel monomer before pouring the stacking gel to ensure continuity between the gels Check pH values of the resolving and stacking gel solu tions Do not back titrate buffers Incomplete gel Allow gel to polymerize fully polymerization 6 p29 problem possible cause Poor band Sample resolution preparation cont Tracking Poor stacking remedy Store sample on ice before it is denatured Dialyze or desalt the sample Heat samples in SDS sample buffer for no more than 1 2 min at 100 C to improve dissociation of subunits Store on ice after heating Adjust the sample volume or concentration Add more mercaptoethanol or dithiothreitol check sam ple treatment Add protease inhibitors such as PMSF if necessary to prevent proteolytic degradation of sample Increase glycerol or sucrose to increase sample density Store samples to be frozen in aliquots to avoid repeated freeze thawing Store at 40 to 80 C Pour a taller stacking gel For best results allow a dye doesn t stacking gel height of 2 5 tim
36. ys install the safety lid before use Glass plates The SE 600 Chroma accommodates 18 cm wide plates 16 or 8 cm long Notched divider plates ordered separately divide gel sandwiches to form club sandwiches of two gels each so up to four gels can be run at one time Clamps Two 16 cm clamps are used to secure the gel sandwich The clamp pressure bar adjusted with screws distributes pressure evenly Casting stand The casting stand holds assembled gel sand wiches upright for casting gels Adjustable feet level the caster laminated gasket in the bottom of each casting cradle seals the bottom of the sandwich when it is clamped into the stand Cams Cams are used twice first to secure the assem bled sandwich in the casting stand and second to attach the sandwich to the upper buffer chamber Rubber gaskets There are two sets of two gaskets The solid laminated gaskets fit into the bottom of the casting stand and form the seal for casting the gel The slotted gaskets fit under the upper buffer chamber and form the seal between the upper and lower chambers The ridges on the upper gasket align the gasket slot to maintain an open channel between the top of the gel and the buffer in the upper chamber Spacers Spacers determine the thickness of the gel and are available in three thicknesses 0 75 1 and 1 5 mm and two widths 1 and 2 cm May be ordered separately Spacer Mate alignment template T
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