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Hoefer DALT System

Loading and Running Second Dimension Gels

Amersham Biosciences 

 

27

Loading and Running Second Dimension Gels

Immobiline DryStrip gels provide a pH gradient (IPG) that is immobilized in an 

acrylamide gel and supported by a plastic film backing. The Hoefer DALT System can 

accommodate the entire length of an 18-cm IPG strip, plus markers, and up to 10 gels 

can be run simultaneously. 

To prepare for second dimension electrophoresis, first equilibrate the focused IPG strips 

in SDS buffer before loading them onto DALT slab gels. 

I

MPORTANT

Prepare the second dimension vertical gel and tank buffer 

before

 IPG 

strip equilibration. Prepare agarose sealing solution during IPG strip 
equilibration.

The exposed edges of the gels tend to dry out over a period of hours. Coordinate both 

IPG and DALT runs so that the end of the IPG run coincides with the end of unloading 

the DALT casting unit. Start the DALT polymerization 2.5 hours before the IPG 

equilibration, to allow up to 2 hours for gel polymerization and 30 minutes for 

unloading the cassettes from the casting unit.

Equilibrating IPG Strips

The SDS equilibration buffer contains urea, glycerol, SDS, Tris buffer and tracking dye. 

During equilibration, the sample proteins are saturated with SDS for mobility in the 

second dimension gel. Urea and glycerol minimize electroendosmosis effects due to the 

IPG strip. In addition, the equilibration buffer contains DTT to fully reduce the sample 

prior to the second dimension separation. 

Equilibration Buffer

 (50 mM Tris-Cl) maintains strip pH in a range that allows proper 

stacking. The pH is typically adjusted to 8.8 to compensate for the acidifying effect of 

the optional iodoacetamide treatment.

Urea  

(6 M), together with glycerol, reduces the effects of electroendosmosis by 

increasing the viscosity of the buffer. Electroendosmosis occurs due to the presence of 

fixed the charges of the IPG matrix in the electric field and can interfere with protein 

transfer from the IPG strip to the second dimension gel.

Glycerol

(30%), together with urea, reduces electroendosmosis and improves transfer 

of protein from the first to the second dimension.

Dithiothreitol (DTT)

preserves the fully reduces state of denatured, unalkylated 

proteins.

Sodium dodecyl sulfate (SDS)

denatures proteins and forms negatively-charged 

protein-SDS complexes. The amount of SDS bound to protein, and therefore the 

additional negative charge, is directly proportional to the mass of the protein. Thus 

electrophoresis of proteins through a sieving gel in the presence of SDS separates 

proteins on the basis of molecular mass.

Содержание Hoefer DALT

Страница 1: ...80 6431 50 Rev A 12 98 Hoefer DALT System DALT Electrophoresis Tank DALT Gradient Maker DALT Multiple Gel Caster DALT Blotting Kit User Manual...

Страница 2: ......

Страница 3: ...ham Biosciences guarantees that the product delivered has been thoroughly tested to assure that it meets its published specifications The warranty included in the conditions of delivery is valid only...

Страница 4: ...producto 1998 Amersham Biosciences Limited Reservados todos los derechos No est permitida la reproducci n ni el almacenaje en un sistema de recuper aci n ni la transmisi n de parte alguna de esta pub...

Страница 5: ...18 Pouring Gel Solutions for Gradient Gels 19 Applying Overlay to DALT Slab Gels 22 Polymerization 22 Unloading the Gel Caster 23 Preparing the DALT Tank for Electrophoresis 24 Connecting a Refrigera...

Страница 6: ...us Gel Solutions 46 Gradient Gel Solutions 49 For 25 gels 1 mm 50 For 12 gels 1 mm 51 For 23 gels 1 5 mm 52 For 12 gels 1 5 mm 53 Gel Identification Numbers 54 Care and Maintenance 55 Cleaning 55 Remo...

Страница 7: ...ple gel caster a gradient maker with peristaltic pump gel cassettes a blotting kit Electrophoresis Tank Figure 1 The DALT Electrophoresis Tank The DALT Electrophoresis Tank accommodates up to ten 23 1...

Страница 8: ...ecomes broken and requires replacement See Removing the Electrode Panels from the DALT Tank on page 55 Figure 2 Rear view of electrophoresis tank Buffer moves into the pump through the middle of the t...

Страница 9: ...n and lifting out the slab The cassette is easily cleaned as a unit and can be stood upright in the open to dry The cassettes are dishwasher safe Cassettes are 20 4 25 5 cm and produce a gel about 19...

Страница 10: ...solution to accommodate the volume shrinkage during polymerization Pump Assisted Gradient Maker Figure 5 The DALT Gradient Maker The DALT Gradient Maker casts gradients of arbitrary shape up to 2 0 li...

Страница 11: ...ing list making sure all items arrived If any part is missing contact your local Amersham Biosciences sales office Inspect all components for damage that may have occurred while the unit was in transi...

Страница 12: ...t doit tre en place avant de brancher les prises au g n rateur Eteindre le g n rateur et d brancher les prises avant d ouvrir le couvercle de s curit Rinser seulement les lectrodes pas les banana plug...

Страница 13: ...amperage 1000 mA Maximum temperature 30 C Environmental operating conditions Indoor use 4 40 C Humidity up to 90 Altitude up to 2000 m Installation category II Pollution degree 2 Buffer circulation p...

Страница 14: ...se 0 40 C Humidity 10 90 Altitude up to 2000 m Installation category II Pollution degree 2 Product certifications EN61010 1 UL508 cUL 115 V IEC 1010 230 V CE DALT Blotting Kit Capacity 5 gels Rack dim...

Страница 15: ...igher current power supply such as the EPS 2A200 is desirable Transfer Membranes The DALT Blotting kit includes 50 pieces of blotting paper For electrophoresis tank blotting Amersham Biosciences offer...

Страница 16: ...ht coating of GelSeal to help assure leak proof sealing Place the gasket in the groove along the front side of the caster Avoid stretching the gasket 3 Start filling the gel caster by placing a separa...

Страница 17: ...d the numbers fall to the floor of the caster but remain in the respective cassettes and ultimately are polymerized into the gels 6 Insert the end of the plastic feed tube supplied with the gel caster...

Страница 18: ...EMED See Homogeneous Gel Solutions on page 46 5 Load the gel caster with cassettes separator sheets and filler blocks if necessary Place a gel label in each cassette See page 12 for directions 6 Conne...

Страница 19: ...er If the V well is not completely filled and the level of gel in the casettes is more than 1 cm below the top of the cassettes you may add up to 50 ml more displacing solution to the balance chamber...

Страница 20: ...g procedure clean all parts of the caster and gradient maker including sponges separating sheets and filler blocks with a solution of mild detergent followed by a fresh water rinse Configuring the Gra...

Страница 21: ...gradient maker Repeat this procedure whenever the required volume of acrylamide solution changes as a result of changing the number or thickness of gels you are casting Calibrating the Peristaltic Pu...

Страница 22: ...The heavy gel solution contains glycerol During the gradient pouring procedure the mixing ratio of heavy solution to light solution gradually increases with the heavier solution underlaying the light...

Страница 23: ...Pouring Gel Solutions for Gradient Gels 1 Prepare the gel caster as described on page 12 placing gel labels in each cassette 2 When you are ready to cast the gels add the APS and TEMED and mix each g...

Страница 24: ...hamber with 150 ml dense displacing solution Figure 12 The grommet seal and gradient feed tube should prevent leakage of displacing solution into the caster Figure 12 Both chambers of the gradient mak...

Страница 25: ...sloped trough at the bottom of the caster If the V well is not completely filled and the level of gel in the casettes is more than 1 cm below the top of the cassettes you may add up to 50 ml more disp...

Страница 26: ...at slab gel tops 1 Immediately after removing the feed tube from the caster slowly deliver 0 75 ml of water saturated n butanol to the surface of each gel The overlay should spread evenly across the c...

Страница 27: ...er with about 0 5 cm of tap water in the bottom The container retains the excess liquid as it drains from the surface of the gel The water in the container helps maintain humidity near the gels A dry...

Страница 28: ...For quick and easy connections install Quick fit connectors with valves in the line 1 Prepare two lengths of vinyl or silicone tubing Slide hose clamps 4 total onto each end of two lengths of tubing...

Страница 29: ...have an airlock in the pump Quickly turn off the pump Wait a moment for the air to bubble out through the small bypass tube that comes up from the pump outlet and enters the back of the tank about ha...

Страница 30: ...is not going to be used for several days siphon out the buffer rinse the tank with water and allow the tank to sit empty This prevents growth of bacteria and molds which can occur in used buffer Such...

Страница 31: ...ring equilibration the sample proteins are saturated with SDS for mobility in the second dimension gel Urea and glycerol minimize electroendosmosis effects due to the IPG strip In addition the equilib...

Страница 32: ...S equilibration buffer see page 43 Just prior to use add DTT to the buffer at a concentration of 100 mg DTT per 10 ml SDS equilibration buffer 2 Place the IPG strips in individual tubes with the suppo...

Страница 33: ...the second dimension gel The edge of the strip should just rest on the surface of the slab gel Avoid trapping air bubbles between the plastic backing and the glass plate or cutting into the SDS gel wi...

Страница 34: ...on Gels Hoefer DALT System 30 Amersham Biosciences Figure 17 Completely cover the IPG strip with agarose sealing solution 4 Keep a log of run conditions and the identification number of the DALT gel o...

Страница 35: ...mmersed in tank buffer Do not drop the plates into the tank and onto the circulation flutes Figure 18 Inserting loaded cassette into DALT Tank 2 Adjust the buffer level after all the cassettes are loa...

Страница 36: ...ration the temperature and pH of the buffers and the number and size of cassettes Observe the progress of your first run and set future schedules accordingly 2 Run the gels until the blue tracking dye...

Страница 37: ...e care not to chip the glass of the cassette When opening the cassette make sure that the gel adheres to one of the plates and is not sticking partly to both to avoid tearing the gel If the gel sticks...

Страница 38: ...s you may add 0 1 SDS and 10 20 methanol Preparing the DALT Tank for Transfer IMPORTANT Gels should be transferred immediately after electrophoresis to avoid sample diffusion Do not soak gels in fixat...

Страница 39: ...fresh buffer add the dry buffer mixture NOTE Even if no cooling is required for your system use the pump to circulate the buffer to avoid buffer depletion at the electrodes Assemble the Transfer Cass...

Страница 40: ...nsfer buffer on the membrane Gently roll a glass pipet or test tube over the gel to expel trapped air between the membrane and gel Cover the gel with a sheet of blotting paper and an additional 6 mm s...

Страница 41: ...kly insert it into one of the sets of vertical slots in the submerged cassette holding rack As many as five gels can be transferred at once in the DALT Blotting Kit For three or less gels use the cass...

Страница 42: ...nt current mode is recommended If constant voltage mode is selected carefully monitor the current Increased current increases Joule heating If the current exceeds 1 0 A decrease the voltage IMPORTANT...

Страница 43: ...te turn the voltage and current settings to zero and turn off the power supply Disconnect the leads from the power supply jacks 2 Open the lid and lift out the cassettes 3 Open each cassette carefully...

Страница 44: ...fects If the sample elution rate is slow a longer transfer period may be required In our experience high current transfers for short periods of time are superior to low voltage transfers for longer pe...

Страница 45: ...900 g Bis N N methylenebis acrylamide purest grade FW 154 17 0 8 24 g Water purest available up to 3000 ml May need filtration Weigh acrylamide and bis under a hood avoid contact with dust Filter and...

Страница 46: ...6 2 10 0 5 ml Water 4 5 ml Prepare fresh in glass vessel Amount Tris Cl 1 5M pH 8 8 50 ml Glycerol 100 ml Bromophenol blue 2 mg Water 50 ml Prepare fresh Stored solution may support microbial growth A...

Страница 47: ...50 mM 6 67 ml Urea FW 60 06 6 M 72 07 g Glycerol 87 v v MW 92 09 30 v v 69 ml SDS FW 288 38 2 w v 4 0 g Bromophenol blue trace a few grains Double distilled H2O to 200 ml Store at 20 C This is a stock...

Страница 48: ...SDS can improve transfer efficiency but may decrease binding to the membrane The pH of this buffer may vary from 8 2 8 4 Do not adjust the pH Adjusting the pH alters the conductivity of the buffer De...

Страница 49: ...ixture into 7 tubes 3 Heat six of the tubes for 4 6 8 10 12 and 15 minutes at 95 C in a heating block or in a boiling water bath After heating place tubes in an ice bucket 4 Mix the contents of the 7...

Страница 50: ...new composition to check that your solution polymerizes in about 10 minutes 1000 ml Amounts shown in ml Final T 7 8 9 10 11 12 13 14 15 16 17 18 19 Acryl Stock 233 267 300 333 367 400 433 467 500 533...

Страница 51: ...15 15 15 15 15 15 15 15 15 15 10 APS 15 00 15 00 15 00 15 50 15 00 15 00 15 00 15 00 15 00 15 00 15 00 15 00 15 00 10 TEMED 3 68 3 21 2 85 2 57 2 33 2 15 1 98 1 83 1 71 1 61 1 52 1 43 1 35 Final T 7 8...

Страница 52: ...8 18 18 18 18 18 18 10 APS 18 00 18 00 18 00 18 00 18 00 18 00 18 00 18 00 18 00 18 00 18 00 18 00 18 00 10 TEMED 4 41 3 85 3 42 3 08 2 79 2 57 2 38 2 20 2 05 1 93 1 82 1 71 1 62 Final T 7 8 9 10 11 1...

Страница 53: ...ions can be degassed though this is not usually necessary Light Solution 1000 ml Amounts shown in ml Heavy Solution 1000 ml Amounts shown in ml Final T 7 8 9 10 11 12 13 14 15 16 17 18 19 Acryl Stock...

Страница 54: ...0 0 0 0 0 0 0 0 0 0 0 0 0 10 APS 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 7 50 10 TEMED 1 84 1 61 1 43 1 28 1 16 11 07 0 99 0 92 0 86 00 80 0 76 0 71 0 68 Final T 8 9 10 11 12 13 1...

Страница 55: ...4 4 4 4 4 4 Glycerol 0 0 0 0 0 0 0 0 0 0 0 0 0 10 APS 4 4 4 4 4 4 4 4 4 4 4 4 4 10 TEMED 0 98 0 86 0 76 0 68 0 62 0 57 0 53 0 49 0 46 0 43 0 40 0 38 0 36 Final T 8 9 10 11 12 13 14 15 16 17 18 19 20 A...

Страница 56: ...9 9 9 9 9 9 Glycerol 0 0 0 0 0 0 0 0 0 0 0 0 0 10 APS 9 9 9 9 9 9 9 9 9 9 9 9 9 10 TEMED 2 21 1 93 1 71 1 54 1 40 1 29 1 19 1 10 1 03 0 96 0 91 0 86 0 81 Final T 8 9 10 11 12 13 14 15 16 17 18 19 20...

Страница 57: ...5 5 5 5 5 5 Glycerol 0 0 0 0 0 0 0 0 0 0 0 0 0 10 APS 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 10 TEMED 1 35 1 35 1 35 1 35 1 35 1 35 1 35 1 35 1 35 1 35 1 35 1 35 1 35 Final T 8 9 10 11 1...

Страница 58: ...phoresed off paper during an SDS electrophoresis run A variety of numbering schemes are possible In our experience the easiest uses three parts as follows An upper case letter to identify the investig...

Страница 59: ...istilled water Allow to air dry Clean glass plates and spacers with a dilute solution of a laboratory cleanser such as RBS 35 from Pierce Chemical Company Rinse thoroughly with tap and distilled water...

Страница 60: ...bis Gel contains swirls If gel polymerized in 10 min too much catalyst Reduce concentration of ammonium persulfate and TEMED by 25 If gel polymerized in 50 min not enough catalyst Increase concentrati...

Страница 61: ...acrylamide solutions and use only stock of the highest quality Only use freshly deionized urea To increase or decrease the migration rate adjust the voltage or current by 25 50 Protein spots are diffu...

Страница 62: ...merged in transfer buffer Gently press on each sponge as it is added to the stack Roll a glass pipette or test tube over the membranes and gel to eliminate all bubbles Process only one strip or membra...

Страница 63: ...red binding surface of the membrane if any contacts the gel Inefficient binding to membrane Chemical parameters Prepare protein transfer buffer without SDS Verify the optimal amount of methanol requir...

Страница 64: ...1945 Dunn M J Corbett J M 1996 2 dimensional polyacrylamide gel electrophoresis Meth Enzymol 271 177 203 Gershoni J M and G E Palade 1983 Protein Blotting Principles and Applications Anal Biochem 131...

Страница 65: ...V 1 80 6068 98 DALT Multiple Gel Caster with filler blocks and separator sheets Cassettes not included 1 80 6330 61 DALT Gel Cassette for 1 0 mm thick gel 25 20 cm 1 80 6067 27 for 1 5 mm thick gel 2...

Страница 66: ...0 mA 1 80 6406 99 EPS 601 Power Supply 600 V 400 mA 1 18 1130 02 PlusOne Electrophoresis Chemicals and Reagents Urea 500 g 17 1319 01 Dithiothreitol DTT 1 g 17 1318 01 Bromophenol Blue 10 g 17 1329 01...

Страница 67: ...vider 16 cooling bath 11 temperature 24 34 creatine kinase charge standards 45 crosslinker concentration 56 culture tubes screw cap 11 D DALT System components 3 to 7 specifications 9 unpacking 7 dega...

Страница 68: ...els 14 solutions 46 O overlay 22 42 57 P peristaltic pump 11 18 polymerization incomplete 56 time 14 15 19 22 power supply current leak 57 electrophoresis 11 proteins contamination 26 35 migration 59...

Страница 69: ...trademarks of Amersham Biosciences Limited or its subsidiaries Amersham is a trademark of Nycomed Amersham plc All other trademarks and registered trademarks are the property of their respective compa...

Страница 70: ...Printed in the USA...

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