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Summary of Contents for Labnet ENDURO VE10
- Page 1 ENDURO™ VE10 Vertical Gel Electrophoresis Systems User Manual ENDURO VE10 System E2010-PA ENDURO VE10 Electroblotting System E2010-PBA Lit #M00608 Copyright 2017 Labnet International, Version # 2...
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Page 2: Table Of Contents
Table of Contents Page Safety Precautions Packing Lists Care and Maintenance Setting Up Gel Casting Gel Preparation Gel Selection Gel Pouring Sample Preparation and Loading Gel Running Blotting Insert Setup Blot Running Appendix Warranty Accessories List... -
Page 3: Safety Precautions
SAFETY PRECAUTION WHEN USED CORRECTLY, THESE UNITS POSE NO HEALTH RISK. HOWEVER, THESE UNITS CAN DELIVER DANGEROUS LEVELS OF ELECTRICITY AND ARE TO BE OPERATED ONLY BY QUALIFIED PERSONNEL FOLLOWING THE GUIDELINES LAID OUT IN THIS INSTRUCTION MANUAL. ANYONE INTENDING TO USE THIS EQUIPMENT SHOULD READ THE COMPLETE MANUAL THOROUGHLY. -
Page 4: Care And Maintenance
Important Notice Care and Maintenance: These products and associated accessories should never come into contact with the following cleaning agents, as these will cause irreversible and accumulative damage: Acetone Chloroform Methanol Carbon tetrachloride Phenol Alkalis Ethanol Isopropyl alcohol Water at temperatures above 60°C will cause damage to the acrylic tanks, trays and other parts. - Page 5 Gel System (E2110-PG-1-BS) Glass thick, 12 sample Plates, Plain with bonded combs 1mm spacers, Pk/2 (E2110-DP) Dummy Plate ENDURO VE10 Electroblotting System Units include tank, lid, internal module, electrodes, Blotting module and include the following accessories: Glass Plates Combs Casting Cooling...
- Page 6 The unit should be checked for damage when received. If damaged, contact carrier and save the box for inspection. Please contact Labnet International if there are any problems or missing items. Usage Guidance and restrictions: •...
- Page 7 Acetone, Phenol, Chloroform, Carbon tetrachloride, Methanol, Ethanol, Isopropyl alcohol, Alkalis. Questions and Service: Should you have a question about the operation of the Spectrafuge Mini Centrifuge or if service is required, contact Corning at: 800-492-1110. Do not send in a unit for service without first calling to obtain a repair authorization number.
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Page 8: Setting Up
4. Refit the lid. The unit is now ready for use. Vertical Gel Casting Using the ENDURO VE10 Gel Casting System:- 1. Clean a set of glass plates for each gel first with distilled water and then with 70% ethanol. One set of glass plates constitutes one notched glass plate and one plain glass plate with bonded spacers. - Page 9 When using a triple glass plate sandwich, the pressure bars will need to be in the completely open position. 4. Position the ENDURO VE10 Gel module on a flat surface. Do not insert the ENDURO VE10 Gel module into the casting base at this point.
- Page 10 Position the ENDURO VE10 Gel insert on the gasket of the casting stand so the key holes are facing the cams. The top of the ENDURO VE10 Gel insert will need to be pushed down very slightly to insert the cams.
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Page 11: Gel Casting
Vertical Gel Casting: 1) Put together bonded 2) Insert inside pressure spacer plain glass plate bar with notched plate with notched plate innermost touching the gasket and module on a flat surface away from the casting base A) Screw Version B) Sliding clamp version 3A) Fully tighten 3B) Fully slide... -
Page 12: Gel Selection
Table 1. ENDURO VE10 Total Gel volume for a 1mm thick gel. For different thicknesses of gel, multiple the below amounts by the spacer thickness. Single – one gel, one dummy plate 7.5 mL Double – two gels 15 mL Using a Triple Plate sandwich –... -
Page 13: Gel Pouring
Table 3: Preparation of the resolving gel solution for two 10 x 10cm gels using 1 mm spacers. Solution 7.50% 17.5% Distilled Water 8.7 mL 7. 5mL 6.3 mL 5.25 mL 3.75mL 2.5 mL 30 % Stock Acrylamide 2.5 mL 3.75 mL 5 mL 6 mL... - Page 14 Table 5. Solution ENDURO VE10 Distilled Water 4.2 mL 30 % Stock Acrylamide 0.65 mL Solution 4 X Stacking Gel Tris- 1.6 mL SDS Solution pH 6.6 10 % Ammonium 67µl Persulphate 7. Carefully mix the stacking gel solution, avoiding generating air bubbles.
- Page 15 Preparation of denatured protein samples for loading: The instructions given below are for denatured samples. For Native samples, please consult a laboratory handbook. 1. Prepare the protein samples for loading. The volume of sample depends on the capacity of the wells. See well chart in the Appendix. 2.
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Page 16: Gel Running
Table 6. Buffer Volume ENDURO VE10 Minimum – Inner tank is filled to above the wells. 250 mL Outer Tank is filled to just flood the bottom of the glass plates. Cooling potential is at a minimum which may affect resolution. - Page 17 6. Release the pressure bars and gently pry apart the glass plates. The gel will usually stick to one of the plates and can be removed by first soaking in running buffer and then gently lifting with a spatula. 7. The gel is now ready to be stained with Coomassie or silver stain for specific band identification and further analysis.
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Page 18: Appendix
Protein Blotting using the ENDURO VE10 Electroblotting Insert Setting up the cassette sandwich: (Common buffer solutions are listed in the Appendix.) 1. Each blot sandwich should be set up as follows:- a. Cassette clamp -ve (black) side placed in a tray or other suitable surface. - Page 19 Note: do not handle the membrane without gloves. 2. Assemble the fiber pads, filter papers, gel and transfer membrane in the above order and roll with a pipette to remove any trapped air. Place on the black and red cassette with the membrane facing the anode (red) side, close the hinge carefully so as to not disturb the sandwich.
- Page 20 5. When the blot time is completed, turn the power supply off and remove the lid of the unit. 6. Remove the cassettes from the main tank. 7. Lift the hinge of each cassette and gently pry apart the blot sandwich and remove the membrane from the gel.
- Page 21 Appendix Stock Solutions for SDS PAGE gels:- Stock 30% Acrylamide Gel Solution:- 30.0 g acrylamide 0.8 g methylene bisacrylamide Distilled Water to 100 mL Stock 4X Resolving Gel Tris (1.5 M Tris HCl pH8.8, 0.4 % SDS) To 110 mL Distilled Water add 36.4 g of Tris base Add 8 mL of 10 % SDS Adjust pH to 8.8 with 1N HCl Adjust the final volume to 200 mL with Distilled Water.
- Page 22 Stock 4X Sample Buffer 4ml glycerol 2ml 2-mercaptoethanol 1.2 g SDS 5ml 4X Stacking Tris 0.03 g Bromophenol blue Aliquot into 1.5ml microcentrifuge tubes. Store at -20°C. Membrane Selection Nitrocellulose Good binding capacity, proteins bind by hydrophobic interactions Pore Size .45µm or 22µm Western Transfer Amino acid analysis...
- Page 23 Buffer Preparation - Proteins Towbin Buffers Native Gels Towbin Buffer pH 8.3 25 mM TRIS, 192 mM glycine, 20% Methanol, 3.0 gm TRIS 14.4 gm glycine 200 ml Methanol add ddi H 2 O to 1 liter Denatured Gels Towbin Buffer pH 8.3, no Methanol 25 mM TRIS, 192 mM glycine 3.0 gm TRIS 14.4 gm glycine...
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Page 24: Warranty
Warranty Statement Corning Incorporated (Corning) warrants that this product will be free from defects in material and workmanship for a period of one (1) year from date of purchase. CORNING DISCLAIMS ALL OTHER WARRANTIES WHETHER EXPRESSED OR IMPLIED, INCLUDING ANY IMPLIED WARRANTIES OF MERCHANTABILITY OR OF FITNESS FOR A PARTICULAR PURPOSE. - Page 25 Accessories Additional accessories available. Contact Labnet International for details. Labnet Cat# Description E2010-BM Blotting Module E2010-B-FB Fiber Blotting Pads, Pack of 6 pads E2010-2D-CT Capillary Tubes, disposable, Pack of 10. E2010-2D-BP Capillary Blanking Ports, Pack of 10 E2010-CP Mini Cooling Pack...
- Page 26 labnetinfo@corning.com www.labnetinternational.com LN135000...
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