Amersham Biosciences Hoefer DALT User Manual Download Page 14

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Hoefer DALT System Function and Description

Hoefer DALT System

Amersham Biosciences

DALT Gradient Maker

Dimensions (h

18 cm

Maximum gradient volume

2,000 ml

Weight

5.4 kg

Peristaltic Pump for Gradient Maker

115 V~

37 W, 1.5A

230 V~

37 W, 0.9A

Weight

4.1 kg (9 lbs.)

Dimensions (h

13.5

22 cm

Environmental operating conditions

Indoor use: 0 – 40 °C
Humidity: 10 – 90%
Altitude: up to 2000 m

Installation category

Pollution degree

Product certifications*

EN61010-1, UL508, cUL (115 V), IEC 1010 (230 V),
CE

DALT Blotting Kit

Capacity

5 gels

Rack dimensions (h

26 cm

Weight

3.2 kg

*This declaration of conformity is only valid for the instrument when it is:

•

used in laboratory locations,

•

used as delivered from Amersham Biosciences, except for alterations

described in the User Manual, and

•

connected to other CE-labeled instruments of products recommended or

approved by Amersham Biosciences.

Summary of Contents for Hoefer DALT

Page 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...

Page 2: ......

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 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...

Page 70: ...Printed in the USA...

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