Electrophoresis Cell

Instruction Manual

For Technical Service Call Your Local Bio-Rad Office or in the U.S. Call 1-800-4BIORAD (1-800-424-6723)

Table of Contents (Summary)

Section 6: Loading the Samples
Section 7: Running the Gel
Section 8: Removing the Gel
Section 9: Maintenance of Equipment
Section 10: Troubleshooting Guide
Section 11: Appendix (Reagents and Gel Preparation, Running Conditions, Staining Comparison)
Section 12: Equipment and Accessories (Mini-PROTEAN II Slab Cell, Accessories, Multi-Casting Chamber, Modular Components, Power Supplies, Chemicals)
Section 13: References

Warranty

Bio-Rad Laboratories warrants the Mini-PROTEAN II cell against defects in materials and workmanship for 1 year. Bio-Rad Laboratories will repair or replace defective parts free of charge during this period. The following defects are specifically excluded:

Defects caused by improper operation.
Repair or modification by unauthorized personnel.
Use of non-Bio-Rad supplied fittings or spare parts.
Damage caused by accident or misuse.
Damage caused by disaster.
Corrosion due to improper solvent or sample use.

This warranty does not apply to platinum wire and glass plates.

For repair service inquiries, contact Bio-Rad Laboratories and provide the model and serial number of the instrument.

1.2 Specifications

Constructions	Molded liquid crystal polymer
Inner cooling core	Glass-filled liquid crystal polymer clamps with acrylic pressure plate
Clamp assemblies	Molded polycarbonate
Lower buffer chamber, lid	Molded polycarbonate
Casting stand	Platinum wire 0.254 mm diameter
Electrodes	Silicone rubber
Gaskets: cooling core, casting stand	Teflon®
Combs	Polyvinyl chloride
Spacers	2.2 kg
Shipping weight	16 cm (L) x 12 cm (W) x 18 cm (H)
Overall size	7 cm (L) x 8 cm (W)
Gel size	(inner) 7.3 cm x 10.2 cm (outer) 8.3 cm x 10.2 cm
Glass plate size	600 VDC
Voltage Limit

Note: Mini-PROTEAN II cell components are not compatible with chlorinated hydrocarbons (e.g., chloroform), aromatic hydrocarbons (e.g., toluene, benzene), acetone, ethanol, or 2-amino-2-methyl-1,3-propanediol. Use of such organic solvents voids all warranties. Contact 1-800-4BIORAD for technical information regarding chemical compatibility.

Safety Standards

This Bio-Rad instrument is designed and certified to meet IEC1010-1 safety standards. Certified products are safe when operated according to the instruction manual. Modification or alteration of this instrument will:

Void the manufacturer's warranty
Void the IEC1010-1 safety certification
Create a potential safety hazard

Bio-Rad is not responsible for injury or damage caused by use of this instrument for unintended purposes or by unauthorized modifications.

*IEC1010-1 is an internationally accepted electrical safety standard for laboratory instruments.

2.1 Sandwich Clamp Assemblies

Sandwich clamp assemblies consist of two clamps with thumbscrews, attached to a clear acrylic pressure plate. A gel sandwich is formed by placing a long (outer) plate against the acrylic pressure plate, adding two spacers, and then a shorter (inner) plate.

The clamp screws and acrylic pressure plate ensure even pressure over the glass plates and spacers, creating a leak-proof seal and preventing plate damage.

2.2 Casting Stand

The casting stand allows two gels to be cast simultaneously. It has one alignment slot and two casting slots. The glass plates and spacers of the gel sandwich are aligned in the alignment slot and then snapped into a casting slot. The rubber gasket provides a leak-proof seal.

Gel Casting Procedure

Step 1: Loosen the four screws on the clamp assembly. Stand it up with screws facing away. Grasp the glass plate sandwich with the longer plate facing away and slide it into the clamp assembly along the front face of the acrylic pressure plate. The longer glass plate should be against the acrylic pressure plate. Tighten the top two screws.

Step 2: Place the clamp assembly into the alignment slot with screws facing away. Loosen the top two screws to allow plates and spacers to settle against the casting stand base. Insert the Mini-PROTEAN II alignment card between the glass plates to position spacers. Tighten both pairs of screws.

Step 3: Remove the alignment card. Pull the sandwich from the alignment slot. Check that plates and spacers are flush at the bottom. Realign if necessary.

Step 4: Level the casting stand with the alignment slot facing you. Ensure the gray silicone gaskets are clean. Place silicone rubber gaskets on the red foam pads of the casting stand slots.

Note: Before transferring to the casting slot, recheck spacer alignment by inverting the gel sandwich. All three surfaces (two glass plates and spacer) must be flush. Misalignment can cause leaks during pouring or running. Repeat alignment if necessary.

Step 5: If casting another gel, align sandwich plates and transfer to the other casting slot. The gel sandwiches are ready for casting.

Stacking Gel Preparation

Step 1: Add APS and TEMED to the degassed monomer solution. Use a pipet and bulb to pour the solution down the spacer nearest the upturned side of the comb. Pour until the bottoms of all teeth are covered. Adjust the comb to its proper position. Add monomer solution to fill the sandwich.

Step 2: Let the gel polymerize for 45 minutes to 1 hour. The gel is ready to load and run. Remove the comb and rinse wells with distilled water.

Section 8: Removing The Gel

After electrophoresis, turn off the power supply and disconnect electrical leads.
Remove the cell lid and carefully pull the inner cooling core out of the lower chamber. Pour off the upper buffer.
Lay the inner cooling core on its side. Remove the clamp assembly by pushing down on both sides of the cooling core latch and up on the clamps until released. Slide the clamp assembly away from the cooling core.
Loosen all four screws of the clamp assembly and remove the glass plate sandwich.
Push one spacer out to the side of the plates.
Gently twist the spacer to pull the upper glass plate away from the gel. The gel will stick to one plate.
Float the gel off the glass plate by inverting the gel and plate under fixative solution or blotting transfer buffer and agitate gently until the gel separates. (See Section 11.5 for fixing and staining protocols.)

Section 9: Maintenance of Equipment

Mini-PROTEAN II cell chamber, inner core, clamps:

Rinse thoroughly with distilled water after every use.

Glass plates, spacers, combs:

Wash with a laboratory detergent (catalog number 161-0722), then rinse thoroughly with distilled water.

Glass plates (for more stringent cleaning):

Soak in a strong acid solution (chromic acid/sulfuric acid cleaning solution) for ≥ 30 minutes and then rinse thoroughly with distilled water.

Warning: Exercise extreme caution for acid cleaning; wear safety glasses, a lab coat, and rubber gloves. Keep a container of NaCO3 nearby to neutralize spills.

Section 10: Troubleshooting Guide

Problem	Possible Cause	Solution
4. Lateral band spreading	a. Diffusion out of the wells prior to turning on the current b. Diffusion during migration through the stacking gel. c. Ionic strength of sample lower than that of gel.	a. Minimize time between sample application and power start up. b. Increase %T of stacking gel to 4.5% or 5% T, or increase current by 25% during stacking. c. Match ionic strength of sample buffer to that of the gel.
5. Skewed or distorted bands.	a. Poor polymerization around sample wells. b. Salts in sample. c. Uneven gel interface.	a. Degas stacking gel solution thoroughly prior to casting; increase ammonium persulfate and TEMED concentrations by 25%; wipe comb teeth with TEMED just before casting. b. Remove salts by dialysis, desalting column, etc. c. Decrease polymerization rate. Overlay gels carefully.
6. Lanes constricted at bottom of gel.	a. Ionic strength of sample higher than that of surrounding gel.	a. Desalt sample and neighboring samples.
12. Leaking during gel casting.	a. Improper assembly of gel sandwich. b. Chipped glass plates.	a. Use alignment card and check that spacers and glass plate bottoms are flush prior to pouring gel. b. Ensure glass plates are free of flaws.
13. Clamp assembly will not fit into casting stand.	a. Incorrectly assembled.	a. Make sure grey and red gaskets are in place. b. Check acrylic block orientation (curved part at bottom). c. Screws should face away from clamp support.

*Polyacrylamide gels are described by reference to two characteristics: 1) The total monomer concentration, (%T) and 2) The crosslinking monomer concentration (%C).

% T = (g acrylamide + g bis-acrylamide) / Total Volume x 100

% C = g bis-acrylamide / (g acrylamide + g bis-acrylamide) x 100

Section 11: Appendix

11.1 Reagents and Gel Preparation for SDS-PAGE Slab Gels (Laemmli buffer system)

B. 1.5 M Tris-HCl, pH 8.8: 27.23 g Tris base (18.15 g/100 ml), ~80 ml deionized water. Adjust to pH 8.8 with 6N HCl. Make to 150 ml with deionized water and store at 4 °C.
C. 0.5 M Tris-HCl, pH 6.8: 6 g Tris base, ~60 ml deionized water. Adjust to pH 6.8 with 6N HCl. Make to 100 ml with deionized water and store at 4 °C.
D. 10% SDS: Dissolve 10 g SDS in 90 ml water with stirring and bring to 100 ml with ddH2O.
E. Sample buffer (SDS reducing buffer): Deionized water (3.8 ml), 0.5 M Tris-HCl, pH 6.8 (1.0 ml), Glycerol (0.8 ml), 10% (w/v) SDS (1.6 ml), 2-mercaptoethanol (0.4 ml), 1% (w/v) bromophenol blue (0.4 ml). Total: 8.0 ml. Dilute sample 1:4 with buffer, heat at 95 °C for 4 minutes.
F. 5X electrode (Running) buffer, pH 8.3: Tris base (15 g/l), Glycine (72 g/l), SDS (5 g/l), to 600 ml with deionized water. Store at 4 °C. Dilute 60 ml 5X stock with 240 ml deionized water for one run.

*Laemmli, U. K., Nature, 227, 680 (1970)

Calculated Volumes Required Per Gel Slab

Spacer Thickness	Volume (ml)
0.5 mm	2.8
0.75 mm	4.2
1.0 mm	5.6
1.5 mm	8.4

*Volume required to completely fill gel sandwich. Amounts may be adjusted.

11.4 Running Conditions

Recommended power condition: 200 volts, constant voltage. No adjustment needed for spacer thickness or number of gels. Usual run time: approximately 45 minutes.

11.5 Comparison of Coomassie Blue and Silver Staining

Procedure	Coomassie Blue	Silver Stain
Staining	Stain 1/2 hour with 0.1% Coomassie blue R-250 in fixative (40% MeOH, 10% HOAc)	As per instructions in kit. 1 hour and 45 minutes for 0.75 mm gel.
Destaining	Destain with several changes of 40% MeOH/10% HOAc to remove background (usually 1 to 3 hr).
Sensitivity	µg range	ng range

Section 12: Equipment and Accessories

12.1 Mini-PROTEAN II® Slab Cell

Components include:

Replacement Red Foam Gaskets, casting stand, 2 (165-2902)
Replacement Gasket, inner cooling core, 2 (165-2905)
Glass Plates, 10 sets* (165-2912)
Inner Glass Plates, 10 (165-2907)
Glass Plates, 10 (165-2908)
Buffer Tank and Lid (165-2975)
Mini-PROTEAN II Electrode Assembly, with gaskets (165-2942)
Replacement Power Cables, 1 pair IEC 1010-1 approved (165-2948)

*One set = one short (inner) plate and one long (outer) plate (enough to cast one gel).

Teflon® Combs

Catalog Number	Product Description
165-2915	5 well x 0.5 mm
165-2916	5 well x 0.75 mm
165-2917	5 well x 1.0 mm
165-2918	5 well x 1.5 mm
165-2919	10 well x 0.5 mm
165-2920	10 well x 0.75 mm
165-2921	10 well x 1.0 mm
165-2922	10 well x 1.5 mm
165-2936	9 well x 0.5 mm
165-2937	9 well x 0.75 mm
165-2938	9 well x 1.0 mm
165-2939	9 well x 1.5 mm

12.3 Mini-PROTEAN II Multi-Casting Chamber and Accessories

Catalog Number	Product Description
165-2950	Mini-PROTEAN II Multi-Casting Chamber, includes casting chamber body with front captive screws, cover with gasket, 10 sets of glass plates, 15 separation sheets, set of 4 acrylic blocks, stopcock valve, luer fittings, and instructions.
165-2913	Replacement Gaskets, Mini-PROTEAN II multi-casting chamber, 3
165-2956	Separation Sheets, 15 sheets
165-2912	Glass Plates, 10 sets
165-2955	Acrylic Blocks, set of 4

12.4 Modular Mini Components for 2-D, Blotting, and Electro-Elution

Catalog Number	Product Description
170-3935	Mini Trans-Blot Module, for use with the Mini-PROTEAN II cell, includes gel holder cassettes (2), fiber pads (4), modular electrode assembly, Bio-Ice® cooling unit, and instruction manual.
165-2960	Mini-PROTEAN II 2-D Cell, includes tube cell module, sample reservoir and stoppers, sample reservoir/capillary tube connectors (50), capillary tubes (200) with casting tube, tube gel ejector, Mini-PROTEAN II slab cell with inner cooling core and gaskets, lower buffer chamber, lid with power cables, glass plates (3 sets),* clamp assemblies (2), 2-D combs with 1 standard well (2), 1.0 mm thick spacers (4), casting stand with gaskets, leveling bubble, and 2-D instruction manual.

12.5 Power Supplies

Model 1000/500 Power Supply, 100/120 V (165-4710)
Model 1000/500 Power Supply, 220/240 V (165-4711)
Model 200/2.0 Constant Voltage Power Supply, 100/120V 50/60 Hz (165-4761)
Model 200/2.0 Constant Voltage Power Supply, 220/240V 50/60 Hz (165-4762)
PowerPac 300 Power Supply, 100/120 V (165-5050)
PowerPac 300 Power Supply, 220/240 V (165-5051)

12.6 Electrophoresis Chemicals

Precast Gels for the Mini-PROTEAN II cell

Catalog Number	Product Description
161-0900	Ready Gel, 7.5% resolving gel, 4% stacking gel
161-0901	Ready Gel, 12% resolving gel, 4% stacking gel
161-0902	Ready Gel, 4-15% gradient gels
161-0903	Ready Gel, 4-20% gradient gels
161-0906	Ready Gel, 10-20% gradient gel, 4% stacking gel
161-0907	Ready Gel, 10% resolving gel, 4% stacking gel
161-0908	Ready Gel, 15% resolving gel, 4% stacking gel
161-0909	Ready Gel, same as 161-0901 with 2 well prep comb

Tris-Borate-EDTA Gels for DNA

Catalog Number	Product Description
161-0904	Ready Gel, 5% TBE gels
161-0905	Ready Gel, 10% TBE gels

Tris-Tricine Gels for Peptides and Small Proteins

Catalog Number	Product Description
161-0922	Ready Gel, 16.5% resolving gel, 4% stacking gel
161-0923	Ready Gel, 10-20% gradient gel, 4% stacking gel

Catalog Number	Product Description	Quantity per Package
161-0755	10x Tris/Glycine/SDS	6 x 1 L
161-0734	10x Tris/Glycine	1 L
161-0757	10x Tris/Glycine	6 x 1 L
161-0733	10x Tris/Boric acid/EDTA	1 L
161-0756	10x Tris/Boric acid/EDTA	6 x 1 L
161-0741	10x TBE Extended Range	1 L
161-0758	10x TBE Extended Range	6 x 1 L
161-0416	10% SDS Solution	250 ml
161-0418	20% SDS Solution	1000 ml
161-0700	Ammonium Persulfate	10 g
161-0800	TEMED	5 ml
161-0801	TEMED	50 ml
161-0710	2-mercaptoethanol	25 ml
161-0611	Dithiothreitol	5 g
161-0404	Bromophenol Blue	10 g

Electrophoresis Stains

Catalog Number	Product Description	Quantity per Package
161-0400	Coomassie Blue R-250	10 g
161-0406	Coomassie Blue G-250	10 g
161-0443	Silver Stain Kit
161-0449	Silver Stain Plus Kit
161-0433	Ethidium Bromide Solution, 10 mg/ml	10 ml

Section 13: References

Laemmli, U. K., Nature, 227, 680 (1970).
Weber, K. and Osborn, M., J. Biol. Chem., 224, 4406 (1969).
Neville, D. M., J. Biol. Chem., 246, 6328 (1971).

Teflon is a registered trademark of E. I. Dupont de Nemours and Company.

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