Bio-Rad PROTEAN Plus Multi-Casting Chamber User Manual

PROTEAN®plus

Multi-Casting Chamber

Instruction Manual

Catalog Number

165-4160

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 Specifications .............................................................................................................1

Section 2 Preparing The Multi-Casting Chamber....................................................2

2.1 Loading the Chamber.................................................................................................2

2.2 Inlet Port Preparation .................................................................................................3

Section 3 Casting Gels ..................................................................................................4

3.1 Casting from the Top (Non-gradient Gels Only).......................................................4

3.2 Casting Gradient Gels ................................................................................................5

Section 4 Removing Gels, Storage and Use of the Gels............................................8

Section 5 Product Information....................................................................................9

Section 6 Reagents for Electrophoresis ......................................................................9

Section 1
General information

1.1 Introduction

The PROTEAN plus Multi-Casting Chamber allows four to twelve 1.0, 1.5 or 2.0 mm gels
to be cast simultaneously for use in the PROTEAN plus Dodeca Cell. Monomer can be
introduced from the top or the bottom to prepare identical linear gradient or single percentage
gels. Cast gels can be stored up to 1 week at 4 ºC.

The PROTEAN plus Multi-Casting Chamber is not used to cast gels for the PROTEAN
II Cell. For this application, use the PROTEAN II xi Multi-Gel Casting Chamber, catalog
number 165-2025.

1.2 Specifications

Construction

Casting chamber fabricated acrylic
Sealing plate fabricated acrylic with captive, spring-loaded

screws

Gasket silicone tubing

Weight 3.9 kg

Approximate size 32cmx23cmx12cm

Gel size 20x20.5(WxL)cmand25x20.5(WxL)cmin

1.0 mm, 1.5 mm, 2.0 mm thicknesses

PROTEAN plus hinged 26.8x22.5cm(WxL)
spacer plate size

Note: PROTEAN plus multi-casting chamber components are not compatible with chlorinated
hydrocarbons (e.g. chloroform), aromatic hydrocarbons (e.g. toluene, benzene), acetone,
glacial acetic acid, carbon tetrachloride, chromic acid (40%), diethyl ether, dimethyl
formamide, ethyl acetate, xylene, and ethanol. Use of such solvents voids all warranties. To
insure best performance of the multi-casting chamber, become fully acquainted with these
instructions before use. All components should be cleaned with a suitable laboratory detergent
(Bio-Rad’s Cleaning Concentrate, catalog number 161-0772), rinsed thoroughly with
distilled water, and dried before use.

1

2

Fig. 1. PROTEAN plus Multi-Casting Chamber.

Section 2
Preparing the Multi-Casting Chamber

2.1 Loading the Chamber

1. Loosen the thumb screws, remove the sealing plate, and place the open casting chamber

face up on the benchtop. The thumbscrew holes should face the ceiling.

2. Start by placing a separation sheet into the chamber so that it seats at the bottom. (Be

sure to remove the protective film from the separation sheet prior to use.)

3. Place the PROTEAN plus hinged spacer plate into the casting chamber body. It is easiest

to insert the plate at a slight angle, hinged side first. Lower the other side into the chamber

so the plate lays flat.

4. Place a separation sheet on top of the hinged spacer plate.

5. Repeat steps 3–4 until you have prepared the desired number of gel sandwiches.

6. Take up the remaining space in the multi-casting chamber with acrylic blocks and

separation sheets so that the sandwiches will be held firmly in position when the sealing

plate is in place.

Note: To insure a good seal, the entire stack should be made as flush as possible to the front
of the multi-casting chamber, and not extend beyond it. If you overfill the multi-casting chamber,
the sealing plate will not be able to create a good seal with the gasket. This causes the monomer
solution to leak during pouring, and glass plates within the stack may break.

Gasket

Thumb screws

Sealing plate

Inlet port with Luer connector

Casting
chamber

Note: Make sure that the plates and separation sheets inside the multi-casting chamber are
seated at the bottom of the multi-casting chamber.

7. Seat the gasket firmly in the notch in the multi-casting chamber body.

8. With the thumb screws loosened, guide the sealing plate into place, being careful not to

disturb the stack. The inlet port should match the groove at the bottom of the multi-

casting chamber. Be careful not to block the inlet port with a separation sheet. Gradually

tighten the screws in a random fashion until the plate is tight.

9. Stand the multi-casting chamber upright on the benchtop, and place it on a level surface.

Do not tip the multi-casting chamber upside-down at this stage.

Note: It may be easier to connect the 6–8 cm piece of tubing (Section 2.2) onto the multi­casting chamber while it is still laying flat on the benchtop, prior to standing it upright.

2.2 Inlet Port Preparation

Casting gels from the TOP

Cut a piece of Tygon tubing (3 mm I.D.) about 6–8 cm in length. Connect one end of the
tubing to the multi-casting chamber port by twisting the tubing side to side until it is on about
1 cm. Attach a stopcock to the other end of the tubing. Close the stopcock valve to prepare for
casting (section 3).

Casting from the BOTTOM

Cut a piece of Tygon tubing (3 mm I.D.) about 6–8 cm in length (a). Connect one end of the
tubing to the multi-casting chamber port by twisting the tubing side to side until it is on about
1 cm. Fit a stopcock to the other end of the tubing (b). Attach the luer connector to the
stopcock (c). Tubing from the gradient former attaches to the luer connector (d). The other end
of the gradient former tubing then is fitted with a luer connector (e), and this fits into a
stopcock (f). This stopcock attaches directly to the gradient former. Close all valves to prepare
for casting (Section 3).

Fig. 2. Tubing and fittings to connect the Multi-Casting Chamber to the Gradient Former.

3

a

b

c

d

e

f

Section 3
Casting Gels

Single percentage gels (non-gradient) can be prepared by introducing monomer from
either the top or bottom of the multi-casting chamber. Gradients must be introduced from the
bottom.

The first time gels of a certain thickness are cast, it is necessary to empirically determine
the required volume of monomer solution. Assemble the stack as outlined in Section 2.1, and
inject a measured volume of water through the stopcock. Prepare this volume (+ 10 ml) of
monomer solution (see sample calculations in Section 3.1 and 3.2). Disassemble the
multi-casting chamber and dry all components.

Note: Wear rubber gloves while performing the following procedure to prevent accidental
exposure to non-polymerized acrylamide, which is a neurotoxin.

3.1 Casting from the Top (Non-gradient Gels Only)

1. Attach a short length of tubing (8 cm of 1/8" ID Tygon®tubing is included) to the inlet

port of the multi-casting chamber. Connect one of the two included stopcocks to the end

of the tubing. Make sure that the valve is in the closed position.

2. Mark a level on the multi-casting chamber (with tape or a pen) at the desired separation

gel length, measuring from the bottom.

3. Prepare the monomer solutions.

Note: For the larger format gels (≥ 20cm in length) it is necessary to decrease the TEMED
amount by 50% (for a final concentration of 0.025%). This will extend the polymerization time
to approximately 20 minutes, providing time to complete casting and for the monomer
solution to settle properly.

Sample Calculation: SEPARATION GEL

Objective: Cast 12 gels, 2.0mm thick, 25 x 20.5 cm, 12% acrylamide.

Place 12 clean hinged spacer plates and separation sheets in the multi-casting chamber
(as described in Section 2.1) and measure the volume required to fill the plates with
water. The volume required to cast the separating gel is approximately 1000 ml (this
volume will vary depending on the desired separating gel height).

Disassemble and dry all components. Prepare 1000 ml of solution. Use the standard
C1V1=C2V2equation.

To prepare 1000 ml (12%)

(12%) (1000 ml) = (X ml) (30% acrylamide/Bis Stock) 400 ml
(0.375M Tris-Cl)(1000 ml) = (X ml) (1.5M Tris-Cl pH 8.8 Stock) 250 ml
(1000 ml) – (400 ml + 250 ml) = ml Water 350 ml
(0.05% APS) (1000 ml) = (X ml) (10% APS Stock) 5 ml
(0.025% TEMED) (1000 ml) = (X ml) (100% TEMED Stock) 250 µl

4. Combine all reagents except the initiators (usually TEMED and APS), and degas the

solution under vacuum for at least 15 minutes. Degassing the solutions removes oxygen

(oxygen inhibits polymerization.)

4

5. After degassing, add initiators to the gel monomer solution, and introduce the solution into

the top of the gel sandwich closest to the sealing plate. Minimize bubble formation

during casting (try to flow the solution down the middle of the sandwich using a large

syringe, or direct flow down the side of the spacer with a pipette or graduated cylinder.)

The groove on the bottom of the multi-casting chamber will equilibrate the solution to each

of the gel sandwiches. Monitor the filling process by observing the level of solution

rising on the sandwich furthest from the sealing plate. Stop when you reach the desired

gel height.

6. Overlay each gel as quickly as possible with 1.0–2.0 ml of water or water-saturated

isobutanol. Optimally, the overlay solution should be applied to every gel simultaneously.

Note: each gel must have the same volume of overlay solution.

7. Allow enough time for complete polymerization of the separating gel before removing the

overlay solution (about 1 hour). While keeping the plates assembled in the chamber, rinse

the gels off thoroughly with water. After rinsing, measure the volume required to fill the

stacking gel area with water. Position the gel on its side and place a rectangular piece of

filter paper between the short and long plates. Dry the stacking gel area, inside the plates

next to the gel, by running the filter paper from top to bottom.

Caution: do not touch or disturb the gel with the filter paper.

8. Prepare the stacking gel monomer solution (see sample calculations below) and apply to

the gels one at a time.

Note: the final concentration of TEMED is 0.1%, the concentration of Tris-Cl stock is

0.5M pH 6.8, and the final concentration is 0.125M.

Sample Calculation: STACKING GEL

Objective: Cast stacking gels for 12 gels, 2.0mm thick, 25 x 20.5 cm.

Measure the volume required to fill the stacking gel area with water. The total
volume required to cast all 12 stacking gels is approximately 350 ml (this volume will
vary according to the height of the separating gel). Prepare 350 ml of solution. Use
the standard equation, C1V1=C2V2.

To prepare 350 ml (4%)

(4%) (350 ml) = (X ml) (30% acrylamide/Bis Stock) 47 ml
(0.125M Tris-Cl)(350 ml) = (X ml) (0.5M Tris-Cl pH 6.8 Stock) 87.5 ml
(350 ml) – (47 ml + 87.5 ml) = ml Water 215.5 ml
(0.05% APS) (350 ml) = (X ml) (10% APS Stock) 1.75 ml
(0.1% TEMED) (350 ml) = (X ml) (100% TEMED Stock) 350 µl

9. Insert a comb into each hinged spacer plate. To minimize bubble formation, insert the

comb at an angle.

Note: You may also cast single percentage gels from the bottom. Use the gradient former, but
just add the single percentage solution into both reservoir chambers.

3.2 Casting Gradient Gels

The inlet port at the bottom of the PROTEAN plus multi-casting chamber is used for
casting linear and convex acrylamide gradient gels using the Model 495 Gradient Former
(catalog number 165-4121). Refer to the gradient former instruction manual for additional
information and for proper operating techniques.

5

Note: GRADIENT GELS ONLY - the size of the V-shaped groove at the base of the multi­casting chamber has been optimized for casting twelve gels. The size of the groove was
optimized so that the monomer solution enters all of the hinged spacer plates at the same
time, rather than one at a time (wicking). However, the larger dimension increases the amount
of monomer solution required to fill the V-groove, thus slightly altering the actual gradient
formed in the gels. With this is mind, TWO solutions are recommended:

• Adjust the heavy solution by approximately 0.5% to help compensate for the
V-groove. For example: If 8–16% linear gradient gels are being cast, prepare 16.5%
acrylamide/Bis for the heavy solution. (see sample calculation below in Section 3.2.2)

• Adjust the amount of light and heavy solution so that they are not equal.

For example: If the total volume required to cast twelve 4–20% gradient gels is 1100 ml,
prepare 555 ml of the light monomer solution (4%) and 545 ml of the heavy monomer
solution (20%). (See sample calculation in Section 3.2.2 and technical directions in
Section 3.2.6)

3.2.1. Place the gradient former on a magnetic stir plate and add a stir bar to the chamber
labeled "light". (See Section 2.2 for inlet port preparation) Place the gradient former
outlet above the top of the plate set, but minimize the length of tubing between it and
the multi-casting chamber.

Fig. 3. Proper set-up to cast 12 gradient gels.

3.2.2. Prepare the heavy and light monomer solutions.

6

Note: For the larger format gels (≥ 20cm in width) it is necessary to decrease the TEMED
concentration by 50% (for a final concentration of 0.025%). This will extend the polymerization
time to approximately 20 minutes, providing time to complete casting and for the monomer
solution to settle properly. Also, adding 10% GLYCEROL to the HEAVY solution is important

when preparing GRADIENT GELS. Glycerol adds "weight" to the heavy solution, the solution
that flows into the multi-casting chamber behind the light solution. This added weight prevents
the heavy solution from pushing up through the middle into the light solution. (This would cause
a ripple in the gradient.) Glycerol is washed out during regular staining and protein digestion
procedures which use acetonitrile and ammonium bicarbonate. It will not interfere with
protein identification (using mass spectrometry).

Sample Calculation: Linear Gradient Gels

Objective: Cast 12 gels, 2.0 mm, 25 x 20.5 cm, 8–16% acrylamide.

Place 12 clean hinged spacer plates and separation sheets in the multi-casting chamber
(as described in Section 2) and measure the volume required to fill the plates with water.
In this example, the volume required is 1330 ml. Disassemble and dry all components.
Prepare 1340 ml of solution, 675 ml of light monomer solution (8%) and 665 ml of heavy
monomer solution (16.5%). Use the standard equation, C1V1=C2V2.

To prepare 675 ml (8%)

(8%) (675 ml) = (X ml) (30% acrylamide/Bis Stock) 180 ml
(0.375M Tris-Cl)(675 ml) = (X ml) (1.5M Tris-Cl pH 8.8 Stock) 169 ml
(675 ml) – (180 ml + 169 ml) = ml Water 326 ml
(0.05% APS) (675 ml) = (X ml) (10% APS Stock) 3.375 ml
(0.025% TEMED) (675 ml) = (X ml) (100% TEMED Stock) 169 µl

To prepare 665 ml (16.5%)

(16.5%) (665 ml) = (X ml) (30% acrylamide/Bis Stock) 366 ml
(0.375M Tris-Cl)(665 ml) = (X ml) (1.5M Tris-Cl pH 8.8 Stock) 166 ml
(10% GLYCEROL) (665 ml) = (X ml) (100% GLYCEROL) 66.5 ml
(665 ml) – (366 ml + 166 ml + 66.5 ml) = ml Water 66.5 ml
(0.05% APS) (665 ml) = (X ml) (10% APS Stock) 3.325 ml
(0.025% TEMED) (665 ml) = (X ml) (100% TEMED Stock) 166 µl

3.2.3. Combine all reagents except the initiators (usually APS and TEMED) and degas the
solutions for 15 minutes under a vacuum. Degassing the solutions removes oxygen
(oxygen inhibits polymerization.)

3.2.4. Immediately prior to pouring, add TEMED and APS to both solutions, mix gently,
and pour the appropriate monomer solutions into the gradient chambers. The light
solution (the one with the lower acrylamide concentration) should be placed in the
mixing chamber labeled "light", and the heavy solution in the reservoir chamber labeled
"heavy". The level of the solutions in the two chambers will NOT be equal at this time.

3.2.5. Turn on the stirring bar in the mixing chamber to a steady speed and maintain this
same speed throughout casting.

Note: If flow rate is kept constant, gradients will be reproducible. Tubing size, the volume of
acrylamide in the chambers, and the rate of stirring must be kept constant. If the rate of
stirring changes, the vortex that is created will also vary. The speed controls the amount of
acrylamide pulled into the mixing (light) chamber from the reservoir (heavy) chamber when
the connection between the two chambers is opened. Variation in stirring can disrupt the
linear gradient formation. Also, the vortex should be off-center inside the mixing chamber.
This can be achieved by moving the gradient former on the stir plate to offset the stir bar.

7

3.2.6. Start casting the gels by opening the STOPCOCK valve to the multi-casting chamber
and the STOPCOCK valve to the gradient former. The valve stem lever on the
gradient former is closed at this time. Allow the light monomer solution to enter the
multi-casting chamber until the level of solution in the mixing chamber is EQUAL to
the level of the heavy monomer solution in the reservoir chamber. The light solution
should be close to entering the plates in the casting chamber. This should help
compensate for the V-groove in the multi-casting chamber.

Note: The gradient must be poured as quickly as possible, without mixing the gradient
solution in the casting chamber. In addition to decreasing the amount of initiators, a peristaltic
pump can be used to pump the entire set of gradients within 10 minutes. However, if it is still
not possible to complete the operation in 10 minutes with a pump, decrease the concentration
of initiators to slow polymerization. An inexperienced user should practice all steps ahead of
time so that the procedure of starting the gradient is completed quickly.

Note: The larger format gels (≥ 20cm in width) require a decrease in the TEMED concentration
by 50% (for a final concentration of 0.025%).

3.2.7. Open the VALVE STEM lever on the gradient former once the levels are EQUAL in
the two chambers, and begin mixing the solutions and creating the gradient. DO NOT
allow any air bubbles to enter the plates.

3.2.8. After casting the gels to the top of the plates, CLOSE the stopcock on the multi­casting chamber and on the gradient former. Carefully remove the luer connector from
the stopcock connected to the multi-casting chamber. Put the tubing into a beaker and
drain the excess acrylamide from the gradient former. Insert the combs at an angle;
this will help minimize bubble formation in the wells.

Caution: Immediately flush the gradient former and tubing with water to prevent
polymerization of residual acrylamide within the gradient former.

Section 4
Removing Gels, Storage and Use of the Gels

Note: Wear rubber gloves while performing the following procedure to prevent accidental
exposure to unpolymerized acrylamide, a neurotoxin.

1. Allow at least one hour for polymerization. After polymerization, remove the sealing

plate from the multi-casting chamber. Remove the gels from the stack one at a time using
the green gel releaser. Place the gel releaser between two plates on the hinged side. Pull
the gel releaser forward, pushing against the back plate still inside the chamber. This
should push and move one plate forward and release it from the rest of the stack. Lift the
hinged spacer plate out of the chamber. Trim off excess acrylamide around the glass
edges with the gel releaser.

Note: Polymerized acrylamide will be between the plates, and adhered to the plates,
separation sheets and edges of the chamber. Use the gel releaser to remove excess acrylamide.

2. Rinse off the tops of all the gels thoroughly with distilled water. Wash off any pieces of

excess acrylamide present with distilled water.

3. Store the gels upright in a tightly sealed container or zip-lock bag. Add a few milliliters

of 1 X gel buffer (identical to the buffer in the gel) to the bottom of the container and to
the tops of the gels to prevent them from drying out. Store tightly sealed at 4 °C.

Note: If a stacking gel is required, it should be cast immediately prior to use.

4. Clean the entire casting chamber thoroughly with distilled water. Residual acrylamide in

the stopcock valve and inlet port can be removed using a pipette tip.

8

Section 5
Product Information

Catalog
Number Description

165-4160 PROTEAN plus Multi-Casting Chamber, includes casting chamber,

sealing plate, silicone gasket, tapered luer connector, leveling
bubble, acrylic blocks and separation sheets. Order plates and combs
separately.

165-4161 Acrylic block (1.5 mm), replacement, 1

165-4162 Acrylic block (3 mm), replacement, 1

165-4163 Acrylic block (6 mm), replacement, 1

165-4164 Acrylic block (12 mm), replacement, 1

165-4165 Separation Sheets,15

165-4170 PROTEAN plus hinged spacer plate, 20 x 20.5 (W x L) cm, 1.0 mm, 1

165-4171 PROTEAN plus hinged spacer plate, 20 x 20.5 (W x L) cm, 1.5 mm, 1

165-4172 PROTEAN plus hinged spacer plate, 20 x 20.5 (W x L) cm, 2.0 mm, 1

165-4173 PROTEAN plus hinged spacer plate, 25 x 20.5 (W x L) cm, 1.0 mm, 1

165-4174 PROTEAN plus hinged spacer plate, 25 x 20.5 (W x L) cm, 1.5 mm, 1

165-4175 PROTEAN plus hinged spacer plate, 25 x 20.5 (W x L) cm, 2.0 mm, 1

165-4176 PROTEAN plus comb, 2-D (1 reference well), 20 cm, 1.0 mm, 1

165-4177 PROTEAN plus comb, 2-D (1 reference well), 20 cm, 1.5 mm, 1

165-4178 PROTEAN plus comb, 2-D (1 reference well), 20 cm, 2.0 mm, 1

165-4179 PROTEAN plus comb, 2-D (1 reference well), 25 cm, 1.0 mm, 1

165-4180 PROTEAN plus comb, 2-D (1 reference well), 25 cm, 1.5 mm, 1

165-4181 PROTEAN plus comb, 2-D (1 reference well), 25 cm, 2.0 mm, 1

165-3320 Gel Releaser,5

Section 6
Reagents for Electrophoresis

Catalog
Number Description

Premixed Electrophoresis Buffers

161-0732 10x Tris/Glycine/SDS Premixed Buffer,1L

161-0733 10x Tris/Boric Acid/EDTA Premixed Buffer,1L

161-0734 10x Tris/Glycine Premixed Buffer,1L

161-0741 10x TBE Extended Range Premixed Buffer,1L

161-0743 50x Tris/Acetic Acid/EDTA Premixed Buffer,1L

161-0744 10x Tris/Tricine/SDS Premixed Buffer,1L

9

Catalog
Number Description

161-0772 10x Tris/Glycine/SDS Premixed Buffer,5L

161-0770 10x Tris/Boric Acid/EDTA Premixed Buffer,5L

161-0771 10x Tris/Glycine Premixed Buffer,5L

161-0758 10x TBE Extended Range Premixed Buffer,6x1L

161-0773 50x Tris/Acetic Acid/EDTA Premixed Buffer,5L

161-0760 10x Tris/Tricine/SDS Premixed Buffer,6x1L

Premixed Sample Buffers

161-0737 Laemmli Sample Buffer,30ml

161-0738 Native Sample Buffer, 30 ml; (Store at 2–8 °C.)

161-0739 Tricine Sample Buffer,30ml

161-0767 Nucleic Acid Sample Buffer, 5X, 10 ml

161-0768 TBE-Urea Sample Buffer,30ml

Acrylamide Solutions

161-0158 30% Acrylamide/Bis Solution 37.5:1, 500 ml

161-0140 40% Acrylamide Solution, 500 ml

161-0142 2% Bis Solution Crosslinker, 500 ml

161-0144 40% Acrylamide/Bis Solution 19:1, 500 ml

161-0146 40% Acrylamide/Bis Solution 29:1, 500 ml

161-0148 40% Acrylamide/Bis Solution 37.5:1, 500 ml

161-0154 30% Acrylamide/Bis Solution 19:1, 500 ml

161-0156 30% Acrylamide/Bis Solution 29:1, 500 ml

Premixed Acrylamide | Bis Powders

161-0123 Acrylamide/Bis 19:1, 150 g

161-0124 Acrylamide/Bis 29:1, 150 g

161-0125 Acrylamide/Bis 37.5:1, 150 g

Premixed Gel Casting Buffers

161-0798 Resolving Gel Buffer 1.5 M Tris-HCl, pH 8.8, 1L

161-0799 Stacking Gel Buffer 0.5 M Tris-HCl,pH6.8,1L

10

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