Hoefer SE 615 and SE 675
Contents
1. Warning Acrylamide is a neurotoxin Wear gloves when handling acrylamide or poly acrylamide Wear a dust mask when weighing acrylamide or preparing acrylamide stocks Pouring standard homogeneous gels Table 4 1 Gel casters and volume required gel caster required volume gradient maker ml size SE 615 190 500 SG 500 SE 675 60 150 SG 100 or SG 500 Actual size depends on calculated volume Q Prepare and degas enough monomer solution to fill all sandwiches to the level of the notched plate The following formula allows extra solution to fill the space between the sandwich and the chamber Monomer vol height x width x spacer x total number of x 1 1 ml cm cm Note The 1 1 multiplier in this formula assures enough solution to fill dead volume thickness cm gel sandwiches No stacking gel Fill solution to just below the top of the notched plate If air pockets form remove with a pipette or syringe Introduce a comb at a slight angle into each sandwich taking care not to trap air under the teeth Stacking gel Fill solution to 4 cm below the top of the rectangular glass plate This height allows 1 cm of stacking gel below the wells Pour the gel and apply an overlay After the gel is set prepare the stacking gel as described on page 15 2 D electrophoresis Fill solution to about 1 0 cm below the top of the rectangular glass plate This height allows 4 to 5 mm of space for th2. parts accessories and related equipment see page 22 Pouring second dimension slab gels When preparing second dimension slab gels in the multiple casters it is important to pour the slabs to the correct height If you are using IPG strips you do not need a stacking gel Pour the second dimension gel to 0 5 1 0 cm below the top of the sandwich for standard casting or 1 cm below the notch if you are casting with divider plates This allows ample room to hold the IPG strips between the two glass plates Use 1 0 or 1 5 mm spacers with IPG strips Fig 1 Components of the SE 615 and 675 Multiple Gel Casters SE 615 and 675 components no description Caster body includes faceplate Spring clamp 4 Foam cord gasket Silicone displacement block Acrylic spacer block Comb Polycarbonate filler sheet Glass plate O WDINIDIaAl R Win yr Spacer jo Inlet port 11 Nylon thumb screw A small silicone displacement block is also available for the SE 675 p4 Fig 2 Building a glass sandwich for caster standard casting glass plate spacer glass plate wax paper or polycarbonate sheet Note Do not block the red inlet port at the bottom of the faceplate Setting up the caster one sandwich Preparing the caster Q If the faceplate is attached to the caste
3. amount of bromophenol blue approximately 75 ml for the SE 615 and 35 ml for the SE 675 Calculate the amount of gradient solution you will need by referring to the formula on page 9 For one dimensional gels Use a 12 cm separation gel with a 4 cm stacking gel For two dimensional gels using IPG strips Calculate the amount of solution needed to fill the sandwiches to approximately 0 5 1 0 cm below the top of the plates Set up the peristaltic pump Using a graduated cylinder and water adjust the flow rate so that the volume of the gradient separation solution plus the volume of the glycerol solution will be completely delivered in 10 15 minutes Q Choose a gradient maker that holds no more than four times the total volume of gradient solution to be poured Check that the gradient maker is clean and that the outlet and mixing port are free of polymerized acrylamide p11 Fig 5 Gradient making system connections p12 Assemble the gradient components See Fig 5 a Close all gradient maker valves and place a stir bar in the mixing chamber the one with the tube connector port b Attach one end of a piece of tubing to the outlet of the gradient maker c Run the other end of the tubing through the peri staltic pump and attach it to a tubing connector d Use a second piece of tubing to attach the tubing connector to the red inlet port at the bottom of the caster mi
4. cross indexing experiments and gel production Appendix B References Laemmli U K 1979 Nature London 227 680 685 SDS PAGE and IEF Handbook p21 p22 Ordering information product qty code no SE 615 Multiple Gel Caster Kit 10 gels 1 SE615 Includes 20 glass plates space saver plate 5 filler sheets 100 sheets of wax paper and Spacer Mate alignment template Order combs and spacers separately SE 675 Multiple Gel Caster Kit 4 gels 1 SE675 Includes 8 glass plates 3 space saver plates 5 filler sheets 100 sheets of wax paper and Spacer Mate alignment template and filler plugs Order combs and spacers separately Foam cord gasket 61 cm x 4 5 mm OD 1 SE208 Red inlet port 4 XPO10 Red spring clamps 4 SE253 Glass plates 18 x 16 cm 2 SE6102 Notched divider glass plates 18 x 16cm 1 SE6102D Acrylic block 11 mm thick 18 x 16 cm al SE612 Polycarbonate filler sheets 5 SEGIS Wax paper precut sheets 18 x 16 cm 100 SE614 Spacer Mate template for aligning spacers 3 SE6119SM Nylon thumb screws 12 SE6003U 2 Silicone filler plug SE 615 1 SE618 Silicone filler plug set SE 675 1 SE678 Includes 1 large and 1 small plug Gel Seal 1 4 oz tube Ki SE6070 product qty code no Related products SE 100 PlateMate plate washer 1 SE100 and storage unit SG 100 Gradient Maker 1 SG100 100 ml total volume SG 500 Gradient maker 1 SG500 500 ml total volume Wonder Wedge T SE1514 Spacers
5. the bottom and sides Place a sheet of wax paper on the polycarbonate sheet Build the first sandwich into the caster Place a sheet of wax paper on top of the sandwich and build the next sandwich Q Repeat step 3 alternating wax paper and gel sand wiches Over the final sandwich lay a polycarbonate sheet instead of wax paper so that the gel solution level is visible through the faceplate Place the faceplate on the caster and make sure the stack fits snugly about 0 5 mm above the edge of the caster If necessary adjust the number of extra poly carbonate sheets acrylic spacer blocks glass plates and sandwiches to obtain a snug fit Make sure the spacers are straight and along the edges of the glass plates Use the Spacer Mate to correct the alignment if necessary If necessary take the faceplate off and make sure all edges of the stack components are flush Press on those that are sticking out until all edges are flush and replace the faceplate on the caster Q Secure the faceplate with the four spring clamps and tighten the bottom thumb screws Casting with divider plates To double the number of gels run simultaneously on the SE 600 and related units place a notched glass divider plate and another pair of spacers between the standard glass plates The result is a club sandwich with two layers of gel formed by the three glass plates To use divider plates follow th
6. BD rama SE 615 amp SE 675 Hoefer SE 615 and SE 675 multiple gel casters gt di J A Atoeter Page finder Introduction aa san enaar sr vanen 1 Gel SIZES anar a aren vr red ae doa An al Homogenous and gradient gels i Standard casting 0 eee eee 2 Casting with divider plates 3 Pouring second dimension slab gels 3 SE 615 and SE 675 components 4 Setting up the caster 2 0000000 5 Preparing the caster 5 Building the stack 6 Casting with divider plates 7 Pouring standard homogenous gels 9 Pouring standard gradient gels 11 Setting up the gradient maker 11 Pouring gradient separation gelS 13 Pouring stacking gels 15 Removing polymerized gels 17 Troubleshooting 0 000 cece eee eee 18 Care and maintenance 2 2 20 Appendix A Gel identification numbers 21 Appendix B References 20055 21 Ordering information 22 Important Most organic solvents including methanol isobutanol isopropanol and even n butanol will craze or cloud the acrylic plastic with prolonged exposure Only use small amounts to overlay the gels pii Safety warnings and precautions Acrylamide is a neurotoxin e Wear gloves when handling acryla
7. add the stacking gel at the last minute to prevent diffusion of buffers between the two gel layers O If you are pouring stacking gels later skip to step 1 under Removing polymerized gels on page 17 If you are pouring stacking gels now calculate the volume of stacking gel monomer solution needed by referring to the following formula Monomer vol height x width x thickness x total number of ml cm cm cm stacking gels where e Height is the distance between the separating gel and the top of the plates e Width is the distance between the spacers Rinse off the water saturated n butanol overlay with distilled water or Tris buffer and invert the caster to drain it Repeat this 2 3 times Degas the stacking gel monomer solution and add catalyst and initiator p15 Note Oxygen inhibits gel polymerization Do not trap air bubbles underneath the comb teeth Note Use gels with stack ing gels immediately Do not store pl6 Q Replace the caster in a vertical position Make sure the surface of the separating gel is free of liquid Use a pipette to fill each sandwich individually with stacking gel monomer solution Hold a comb attached to a comb back at a 45 angle and centered within the sandwich Insert one end into the gel so that the end tooth is almost completely inserted Slowly lower the remaining comb teeth in one by one rotating the comb gradually d
8. e IPG strip and an agarose seal Overlay the separating gel as described in step 3 Caution sobutanol crazes or clouds the acrylic caster walls making it difficult to see the gels plo Add initiator and catalyst and pour the solution into the glass sandwiches from the top The solution flows from one sandwich to another through the groove at the bottom of the caster and rises to the same level in all sandwiches Pour slowly to avoid overfilling After reaching the final level overlay each gel sandwich with 300 ul of water saturated n butanol or buffer Use the same amount of overlay on each gel sandwich to assure that all the gels polymerize to the same heights Pour the liquid gently taking care not to disrupt the gel surface After polymerization is complete remove the n butanol and replace it with buffer or add stacking gel Water saturated n butanol will cloud acrylic parts if used in large amounts or for long periods Q Allow the gels to polymerize for at least an hour To remove the polymerized gels see Removing polym erized gels on page 17 To add a stacking gel see Pouring stacking gels on page 15 Pouring standard gradient gels See Fig 1 on page 4 for a diagram of the caster components See Setting up the caster on page 5 for recommended solution volumes Setting up the gradient maker Prepare a 50 glycerol displacement solution containing a small
9. e directions in Pouring standard homogeneous gels on page 9 or Pouring standard gradient gels on page 11 When calculating solution volumes use the formula on page 9 When stacking glass sandwiches use the following procedure Q Stack glass sandwiches in the caster using one club sandwich for each two gels you will cast Fill extra space with acrylic blocks glass plates and polycar bonate sheets Slide in one polycarbonate sheet against the back Slide in a glass club sandwich See Fig 3 To construct a club sandwich slide in one rectangular glass plate and place spacers along each of the two side edges Slide in a notched divider glass plate and place spacers along each of the side edges Finally slide in a second rectangular glass plate Note For optimum visibility place a polycarbonate sheet against the faceplate Fig 3 Constructing a club sandwich for casting with divider plates glass plate spacer notched divider plate spacer glass plate wax paper or polycarbonate sheet Q Place a polycarbonate sheet or wax paper on top of the club sandwich Repeat step 3 alternating wax paper and club sandwiches After the last club sandwich add acrylic spacer blocks and extra polycarbonate sheets until the entire stack is approximately 0 5 mm above the rim of the box one club sandwich additional club sandwiches
10. e the gels p14 When almost all the acrylamide solution is drained from the gradient maker stop the pump and close the mixing valve Tilt the gradient maker towards the outlet side and remove the last few milliliters of mix Do not allow any air bubbles to enter the tubing O Add the glycerol displacement solution to the mixing chamber and start the pump Make sure no bubbles are introduced Pump until the bottom of the caster is filled with displacement solution to just below the glass plates then turn off the pump Clamp off the tubing to the red inlet port of the caster o Overlay each separate sandwich with 300 ul of water saturated n butanol or buffer Use the same amount of overlay on each gel to assure that all the gels polymerize to the same height Carefully and gently pipette the liquid taking care not to disrupt the gel surface After polymerization remove the n butanol Replace it with buffer or add a stacking gel Allow the gels to polymerize for at least an hour To remove the polymerized gels see Removing polymerized gels on page 17 To add a stacking gel see Pouring stacking gels on page 15 a Pouring stacking gels You may add stacking gels to the entire set of polymerized gels homogeneous or gradient while the sandwiches are still in the casting box or you may add stacking gel to individual gels immediately prior to use For optimal resolution
11. length thickness width cm mm cm 16 0 79 2 2 SE6119 2 75 16 1 0 2 2 SE6119 2 1 0 16 1 5 2 2 SE6119 2 1 5 16 1 0 1 2 SE6118 2 1 0 16 1 5 1 2 SE6118 2 1 5 Combs Teflon Combs for the SE 615 and SE 675 are available for the slab thicknesses 0 75 1 0 and 1 5 mm with 28 20 15 12 and 10 wells Blank combs and single reference well combs are also available Please see the Hoefer catalog under SE 600 accessories p23 Hoefer Inc 953 Indiana Street San Francisco CA 94107 USA www hoeferinc com Teflon is a trademark of E duPont de Nemours and Co 2004 Hoefer Inc All rights reserved Printed in the USA Hoefer
12. mide or polyacrylamide e Wear a dust mask when weighing acrylamide or preparing acrylamide solutions Protecting your equipment To keep your instrument in excellent condition please take the following important steps e Use water saturated n butanol for overlayering resolution gels during polymerization e Avoid using small metal spatulas to separate the plates Spatulas may chip the edges of glass plates and prevent them from sealing After casting you may need to use a wedge to separate one glass and gel sandwich from another Slip a Wonder Wedge in between adjacent sandwiches to separate them Introduction Gel sizes The Hoefer SE 615 and SE 675 Multiple Gel Casters are designed for casting vertical slab acrylamide gels of homogenous or gradient concentration Use the casters with 16 x 18 cm glass plates and 2 cm wide spacers to cast 16 x 14 cm gels Use 1 cm wide spacers to cast 16 x 16 cm gels for two dimensional electrophoresis The resulting gels are compatible with Hoefer SE 400 SE 600 and SE 600 Chroma Vertical Slab Gel Electro phoresis Units Homogeneous and gradient gels The SE 615 and SE 675 can be used for casting both homogeneous and gradient gels Gradient gels are cast through the port at the bottom of the caster When casting homogeneous gels the results are most consistent when the gels are cast from the bottom The V shaped region at the bottom of the casting unit allows the gel solu tion t
13. nse the individual sandwiches with distilled water Fill cassettes with gel storage buffer Wrap gel sand wiches you are not using at this time in plastic wrap and store them in the refrigerator To use gels immediately O Gently remove the combs by pulling them upward and out of the gel sandwich Fill the wells with SDS Electrophoresis buffer Load samples and complete assembly Refer to the user manuals that come with SE 600 or SE 400 Series electrophoresis systems p17 Troubleshooting symptom possible cause recommended action 1 Gels adhere to glass plates when opening sandwich 2 Gels cast simultaneously are different sizes 3 Caster leaks 4 Gel heights uneven 5 Sample wells damaged or leak 6 Gel sandwiches difficult to separate 7 Uneven gradient gels p18 Dirt grease or fingerprints on plates Different amounts of overlay were used on the separation gels before polymerization Gasket leaks Gasket damaged Stack too tall Bubbles trapped under gel sandwiches Polymerized gel in groove Insufficient time for gel levels to stabilize Air bubbles under comb teeth Insufficient polymerization Comb removed too abruptly Poor polymerization No wax paper between glass plates Uneven layering Soak plates in a strong laboratory detergent rinse well in distilled water Handle with gloves only Use the same amou
14. nt of overlay on all separation gels Add the overlay as rapidly as possible Apply a light film of Gel Seal to the gasket each time the unit is used Check the foam gasket for nicks or wear and replace if necessary Remove filler plates or gel sandwiches until the stack top is just below the level of the caster wall Pour the monomer solution into one sandwich and allow the groove in the plug to evenly distribute the solution Make sure the groove in the triangular plug is clean and clear of material Wait one minute before overlaying the gels Add the same amount of overlay to each sandwich Remove air pockets before inserting combs Slide comb into solution at an angle If comb must be removed add more monomer solution before reinserting the comb Allow the gel to set for a minimum of 1 hour Slowly remove the comb at a slight angle to prevent damaging the gel Degas stacking gel solution Increase catalysts up to 0 1 v v TEMED 0 1 v v APS Place wax paper between sandwiches in the stack Add sucrose or glycerol to the heavy monomer solution Add a small amount of bromophenol blue to the heavy solution to track gradient formation Decrease the pump rate symptom possible cause recommended action 8 9 10 11 No polymerization of SDS gel or incomplete polymerization Gel too soft Gel is brittle Gel is white Gel contains swirls polymerization artifacts Bands are diff
15. o rise evenly producing identical gels Standard casting In the standard casting procedure a gel is cast between two glass plates held apart by a pair of spacers See Fig 2 on page 5 The result can be pictured as a sandwich with the gel between two glass plates When you set up a vertical slab gel electrophoresis unit you place the glass and gel sandwich in the electrophoresis unit The maximum number of gels you can cast simultaneously using the standard casting proce dure is shown in Table 1 1 Table 1 1 Maximum number of standard gels cast 0 75 mm 1 0 mm 1 5 mm spacers spacers spacers SE 615 11 10 10 SE 675 4 4 4 Casting with divider plates You may find it useful to double the number of gels run simultaneously in one electrophoresis unit This can be accomplished by casting a club sandwich A club sandwich contains a notched glass divider plate and a second pair of spacers between the standard plates and thus forms two layers of gel between three glass plates see Fig 3 on page 8 Divider plates are available for the SE 400 and SE 600 The maximum number of gels you can cast simultaneously using divider plates two gels per sandwich is shown in Table 1 2 Table 1 2 Maximum number of gels cast with divider plates 0 75 mm 1 0 mm 1 5 mm spacers spacers spacers SE 615 14 14 12 SE 675 6 6 4 See Fig 1 on page 4 to identify the components of the multiple gel casters For information on
16. ons See 8 Do not store liquid acrylamide more than 3 months Use gels within 1 2 weeks of casting Use gels with stackers immediately Degas gel solutions at least 10 minutes Use reagents specified as electrophoresis purity Use only double distilled water p19 p20 Care and maintenance e Do not autoclave or heat any part above 45 C e Do not expose the caster or its parts to organic solvents Cleaning Rinse the caster faceplate silicone plugs and polycarbonate sheets in dilute detergent and rinse with distilled water Allow the unit to air dry completely Appendix A Gel identification numbers For positive identification of gels label each slab by incorporating a small label printed on thin filter paper in the bottom corner of the gel Use a carbon typewriter ribbon photocopier or laser printer to make these labels since many liquid based inks are electrophoresed off paper during an SDS electrophoresis run A variety of numbering schemes are possible In our experience the easiest uses three parts as follows e An upper case letter to identify the investigator or an extended gel series e A two or three digit serial number to identify the slab gel batch e A lower case letter to identify a gel in the batch Since a maximum of 14 gels can be made in a batch use the letters a n The resulting numbers in the format A63a A63b etc provide a useful system for keep ing track of and
17. ownward until it is in a horizontal position Refer to Fig 1 on page 4 for the final position of the comb Repeat this for each sandwich Allow the stacking gels to polymerize for at least one hour To remove the polymerized gels see Removing polymerized gels on page 17 Note The SDS buffer makes the glass plates slippery Note Do not use metal spatulas to separate the sand wiches Narrow metal spatulas often chip the edges of glass plates making the plate ineffective for sealing into the electrophoresis tank Note Separate the sandwiches from one another before plac ing the gels in the refrigerator Removing polymerized gels Q After the gels have polymerized place the caster in a horizontal position If you poured stacking gels leave the combs in place Remove the faceplate Tip the caster to pour off the glycerol solution Slide the stack of sandwiches acrylic blocks and polycarbonate sheets out of the caster Remove the acrylic blocks and polycarbonate sheets from the top of the stack Q Carefully remove individual glass and gel sandwiches from the stack If necessary insert a Wonder Wedge between adjacent sandwiches to separate them Work slowly and cautiously when prying apart adjacent sandwiches after polymerization Rinse the caster and polycarbonate sheets as described in Care and maintenance on page 20 To store gels for future use Ri
18. r remove it Remove the four side clamps loosen the two thumb screws and slide off the faceplate Remove the foam cord gasket Apply a light coat of Gel Seal lubricant and replace the gasket When casting gradient gels attach the tubing from the gradient maker to the inlet port at the bottom of the faceplate Homogeneous gels may also be cast through this port If casting homogeneous gels from the top insert the appropriate displacement block in the open V shaped region at the bottom of the chamber 4 Place the open caster in a horizontal position with its back on the bench top Note To make separation of polymerized gel sandwiches easier place wax paper sheets in the caster between each set of glass sandwiches Note When analyzing a large number of gels you may find it useful to include gel iden tification numbers into the polymerized gel See the appendix on page 21 for directions on making gel identification tags Lightly wet and place the tags in the lower corner of the gel sand wich during stack assembly Note If you use polycarbonate sheets instead of wax paper between each sandwich the caster holds fewer sandwiches Building the stack O Place a polycarbonate sheet in the caster so that 1 3 of the sheet extends out of the top You can use this sheet as a lever when inserting filler sheets after all sandwiches are in place All remaining components fit flush against
19. use or broad Protein mobilities not consistent Heavy background during silver staining Insufficient APS or TEMED APS stack is wet 02 in gel solution Solutions at low temperature TEMED is old Not enough crosslinker Too much bis Too much bis Too much catalyst gel polymerized in lt 10 min Not enough catalyst gel polymerized in gt 50 min Solutions not mixed Sample doesn t contain same buffer as stacking gel Too much TEMED or APS SDS or sample buffer is old Poor interface between separation gel and stacking gel Incomplete polymerization Aged gels or acrylamide Gas in gel Acrylamide or bis contain acrylic acid Water is impure Increase both APS and TEMED by 30 50 APS solution is old Make up fresh APS each day APS is hygroscopic Open a fresh bottle Degas at least 10 minutes Make sure all solutions are at room temperature 20 30 C Use new TEMED Crosslinker should be 2 6 C for standard SDS gels where Ce _Sbis _ g monomer g bis See 9 above Check concentrations of solutions See 9 above Reduce both APS and TEMED by 25 x 100 Increase both APS and TEMED by 50 Also see 8 Mix thoroughly after adding TEMED Use the same buffer for the sample as for the stacking gel Reduce concentrations by 25 Use fresh solutions Remove all liquid from the surface of the separation gel before adding the stacking gel soluti
20. xing chamber reservoir chamber multiple gel gradient maker caster ae peristaltic pump magnetic stirrer Warning Acrylamide is a neurotoxin Wear gloves when handling acrylamide or poly acrylamide Wear a dust mask when weighing acrylamide or preparing acrylamide stocks Pouring gradient separation gels Q Prepare and degas the two monomer solutions for the gradient maker Add glycerol or sucrose to the solution of higher acrylamide concentration to stabilize the gradient as the solution is pumped in Decrease the concentration of initiator so that polymerization occurs from top to bottom This minimizes convective mixing due to the heat generated by polymerization Close both the mixing port and outlet port if appropriate on the gradient maker Clamp the tubing from the outlet when using the SG 500 Add initiator and catalyst and immediately pour the light low concentration acrylamide solution into the mixing chamber the chamber with the outlet port Open the mixing valve slightly to allow the connecting channel to fill and force out air bubbles Close the valve again and pour the heavy high concen tration acrylamide solution into the reservoir chamber Q Start the magnetic stirrer and unclamp or open the outlet valve Start the pump and open the mixing valve p13 Note sobutanol clouds the acrylic making it difficult to se
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