Bio-Rad Mini-Protean 3 Cell User manual

Mini-PROTEAN®3 Cell

Instruction Manual

Catalog Numbers

165-3301
165-3302

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

Page

Section 1 General Information....................................................................................1

1.1 Introduction ................................................................................................................1

1.2 Components................................................................................................................2

1.3 Specifications .............................................................................................................4

1.4 Chemical Compatibility .............................................................................................4

1.5 Safety ..........................................................................................................................5

Section 2 Set Up and Basic Operation........................................................................5

2.1 Gel Cassette Preparation ............................................................................................5

2.2 Mini-PROTEAN 3 Cell Assembly and Sample Loading .........................................8

2.3 Gel Electrophoresis ....................................................................................................9

Section 3 Separation Theory and Optimization......................................................10

3.1 Introduction ..............................................................................................................10

3.2 SDS-PAGE (Laemmli) Buffer System....................................................................11

3.3 Native PAGE............................................................................................................12

Section 4 Reagent Preparation and Stock Solutions ..............................................13

4.1 Volumes Required Per Gel ......................................................................................13

4.2 SDS-PAGE (Laemmli) Buffer System....................................................................13

4.3 Discontinuous Native PAGE (Ornstein-Davis).......................................................15

4.4 Continuous Native PAGE ........................................................................................16

Section 5 References ...................................................................................................18

Section 6 Maintenance ...............................................................................................18

Section 7 Troubleshooting..........................................................................................19

Section 8 Product Information and Accessories .....................................................21

Section 9 Warranty Information...............................................................................23

Section 1
General Information

1.1 Introduction

The Mini-PROTEAN 3 cell runs both hand cast gels and Ready Gel precast gels
interchangeably. The Mini-PROTEAN 3 system includes a casting stand and glass plates with
permanently bonded gel spacers that simplify hand casting and eliminate leaking during
casting. The cell can run one or two gels, and the mini tank is compatible with other Bio-Rad
electrode modules for tank blotting, 2-D electrophoresis, and electro-elution.

Fig. 1. Mini-PROTEAN 3 system components.

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2

1.2 Components

To get the best performance from your Mini-PROTEAN 3 cell, familiarize yourself with
the components by assembling and disassembling the cell before using it (refer to
Figures 1 and 2).

Spacer Plate The Spacer Plate is the taller glass plate with gel spacers

permanently bonded. Spacer Plates are available in 0.5 mm, 0.75 mm,

1.0 mm, and 1.5 mm thicknesses, which are marked directly on
each Spacer Plate.

Short Plate The Short Plate is the shorter, flat glass plate that combines with the

Spacer Plate to form the gel cassette sandwich.

Casting Frame The Casting Frame, when placed on the benchtop, evenly aligns

and secures the Spacer Plate and the Short Plate together to form the
gel cassette sandwich prior to casting.

Gel Cassette Assembly One Casting Frame, a Spacer Plate, and a Short Plate form one Gel

Cassette Assembly.

Casting Stand The Casting Stand secures the Gel Cassette Assembly during gel

casting. It contains pressure levers that seal the Gel Cassette
Assembly against the casting gaskets.

Gel Cassette Sandwich A Spacer Plate and Short Plate with polymerized gel form a Gel

Cassette Sandwich after casting.
Combs A selection of molded combs is available.
Buffer Dam The molded, one-piece buffer dam is used when running only one

gel.
Electrode Assembly The Electrode Assembly holds the Gel Cassette Sandwich. It houses

the sealing gasket, the upper and lower electrodes and the

connecting banana plugs. The anode (lower electrode) banana plug

is identified with a red marker and the cathode (upper electrode)

banana plug with a black marker.
Clamping Frame The Clamping Frame holds the Electrode Assembly and Gel

Cassette Sandwich in place. Its pressure plates and closure cams

seal the Gel Cassette Sandwich against U-shaped gaskets on the

Electrode Assembly to form the inner buffer chamber.
Inner Chamber The Electrode Assembly, two Gel Cassette Sandwiches or one gel

cassette sandwich and a buffer dam, and the Clamping Frame form

the Inner Chamber.
Mini Tank and Lid The Mini Tank and Lid combine to fully enclose the inner chamber

during electrophoresis. The lid cannot be removed without

disrupting the electrical circuit. The Mini Tank and Lid are also

compatible with other Bio-Rad electrode modules for blotting, first

dimension 2-D, and electro-elution.

Fig. 2. Assembling the Mini-PROTEAN 3 cell.

Fig. 3. Assembling the Mini-PROTEAN 3 Casting Frame and Casting Stand.

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Lid

Electrode
Assembly

Clamping
Frame

Mini Tank

Spring loaded levers

Casting Stand without
gaskets. Gaskets must be
used for proper seal.

Casting
Frame

Pressure cam
pivot point

Pressure cams in
"open position"

Inner
Chamber
Assembly

Cams

Pressure Plate

Gel Cassette

Sandwich

Notch

on U-Shaped Gasket

Banana Plugs

Anode banana plug

(red)

Cathode banana plug
(black)

1.3 Specifications

Casting Stand* Polycarbonate

Pin, Retaining Ring, and Spring Stainless Steel

Casting Frames* Polysulfone

Gray Gaskets Silicone Rubber (gray)

Clamping Frame** Glass-filled liquid crystal polymer (Vectra

™

)

Pressure Plate and Cams Polycarbonate

Electrode Assembly Glass-filled liquid crystal polymer

Electrodes Platinum wire, 0.010 inches diameter

Gasket, electrode inner core Silicone Rubber (green)

Mini Tank and Lid Molded Polycarbonate

Sample Loading Guides

†

Delrin

™

Combs* Polycarbonate

Maximum Sample Volume Per Well

# wells Well width 0.5 mm 0.75 mm 1.0 mm 1.5 mm
5 12.7 mm — 70 µl 105 µl 160 µl

9 5.08 mm — 33 µl 44 µl 66 µl

10 5.08 mm 22 µl 33 µl 44 µl 66 µl

15 3.35 mm 13 µl 20 µl 26 µl 40 µl

IPG 76.2 mm — — 420 µl 730 µl

Prep/2-D

Reference well 3.1 mm — 13 µl 17 µl 30 µl
Sample well 71.7 mm — 310 µl 400 µl 680 µl

Overall Size of cell 16cm(L)x12cm(W)x18cm(H)

Gel Size 8 cm (W) x 7.3 cm (H)

Inner Plate 10.1 cm (W) x 7.3 cm (H)

Outer Plater 10.1 cm (W) x 8.3 cm (H)

Precast Gel Compatibility Ready Gels

Voltage Limit 600 VDC and 15 watts

Shipping Weight 2.0 kg

1.4 Chemical Compatibility

Mini-PROTEAN 3 components are not compatible with acetone, ethanol, or butanol. Use of
organic solvents voids all warranties. Call 1-800-4-BIORAD or your local Bio-Rad
representative for technical information regarding additional chemical compatibility of the
Mini-PROTEAN 3 cell with various laboratory reagents.

The Mini-PROTEAN 3 combs are not compatible with repeated exposure to 100%
TEMED. Rubbing the combs with TEMED prior to casting will destroy the structural
integrity of the combs over time.

* US patent No. 6,162,342

** US patent No. 5,632,877

†

US patent No. 5,656,145

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1.5 Safety

Power to the Mini-PROTEAN 3 cell is supplied by an external DC voltage power supply
(not included). The output of this power supply must be isolated from external ground to
insure that the DC voltage output floats with respect to ground. All Bio-Rad power supplies
meet this important safety requirement. Regardless of the power supply used, the maximum
specified operating parameters for the Mini-PROTEAN 3 cell are as follows:

• 600 VDC maximum voltage limit

• 15 watts maximum power limit

• 50 °C maximum ambient temperature limit

The current to the cell enters the unit through the lid assembly which provides a safety
interlock to the user. The current to the cell is broken when the lid is removed. Always turn
off the power supply before removing the lid. Do not attempt to use the cell without the

safety lid.

Important: This Bio-Rad product is designed and certified to meet *EN61010-1 safety

standards. Certified products are safe to use when operated in accordance with the instruction
manual. This instrument should not be modified or altered in any way. Alteration of this
instrument will

• Void the warranty

• Void the EN61010-1 certification, and

• Create a potential safety hazard.

Bio-Rad is not responsible for any injury or damage caused by use of this instrument for
purposes other than those for which it is intended or by modifications of the instrument not
performed by Bio-Rad or an authorized agent.

*

EN61010-1 is an internationally accepted electrical safety standard for laboratory instruments.

Section 2
Set Up and Basic Operation

2.1 Gel Cassette Sandwich Preparation

Hand Cast Gels

1. Glass Cassette and Casting Stand Assembly

Note: Ensure the casting stand, casting frames, and glass plates are clean and dry before

setting up the casting stand assembly. During regular use, a powder residue may build

up behind the pressure cams of the casting frame at the pivot point. This powder should

be removed before each use.

a. Place the Casting Frame upright with the pressure cams in the open position and

facing forward on a flat surface.

b. Select a Spacer Plate of the desired gel thickness and place a Short Plate on top of

it (see Figure 4a).

c. Orient the Spacer Plate so that the labeling is "up". Slide the two glass plates into the

Casting Frame, keeping the Short Plate facing the front of the frame (side with
pressure cams) (see Figure 4b).

Note: Ensure both plates are flush on a level surface and labeling on the Spacer Plate is

oriented correctly. Leaking may occur if the plates are misaligned or oriented incorrectly.

d. When the glass plates are in place, engage the pressure cams to secure the glass

cassette sandwich in the Casting Frame (see Figure 4c). Check that both plates are
flush at the bottom.

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e. Engage the spring loaded lever and place the gel cassette assembly on the gray cast-

ing stand gasket. Insure the horizontal ribs on the back of the Casting Frame are
flush against the face of the Casting Stand and the glass plates are perpendicular to
the level surface. The lever pushes the Spacer Place down against the gray rubber
gasket (see Figure 4d).

f. Repeat steps a–e for a second gel.

4a. Place a Short Plate on top of the 4b. Slide the two plates into the Casting
Spacer Plate. Frame keeping the Short Plate facing front.

4c. Lock the pressure cams to secure 4d. Secure the Casting Frame in the Casting
the glass plates. Stand by engaging the spring loaded lever.

Fig. 4. Assembling the Mini-PROTEAN 3 casting stand and frame.

2. Gel Casting

a. Discontinuous Polyacrylamide Gels

i. Place a comb completely into the assembled gel cassette. Mark the glass plate

1 cm below the comb teeth. This is the level to which the resolving gel is
poured. Remove the comb.

ii. Prepare the resolving gel monomer solution by combining all reagents except

APS and TEMED. (Refer to Section 4 for gel formulations.) Degas the
solution under vacuum for at least 15 minutes. Do not use a sink water aspirator.

iii. Add APS and TEMED to the degassed monomer solution and pour to the mark

using a glass or disposable plastic pipette. Pour the solution smoothly to
prevent it from mixing with air.

iv. Immediately overlay the monomer solution with water or t-amyl alcohol.

Note: If water is used, add it slowly and evenly to prevent mixing. Do not overlay

w/butanol or isobutanol.

v. Allow the gel to polymerize for 45 minutes to 1 hour. Rinse the gel surface

completely with distilled water. Do not leave the alcohol overlay on the gel
for more than 1 hour because it will dehydrate the top of the gel.

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