Bio-Rad 491 Prep Cell and Mini Prep Cell User Manual
Model 491 Prep Cell
Instruction Manual
For Technical Service call your local Bio-Rad office or in the U.S. call 1-800-424-6723.
Note
To ensure the best performance from the Model 491 prep cell, become fully acquanted with these operating
instructions before using the cell to transfer samples. Bio-Rad recommends that you first read these instructions
carefully. Then assemble and disassemble the cell completely without transferring sample. After these preliminary steps, you should be ready to transfer a sample.
Bio-Rad also recommends that all Model 491 prep cell components and accessories be cleaned with a suitable
laboratory cleaner (such as Bio-Rad cleaning concentrate, catalog number 161-0722) and rinsed thoroughly
with distilled water, before use.
Model
Catalog No.
Date of Delivery
Serial No.
Invoice No.
Purchase Order No.
Warranty
Bio-Rad Laboratories’ Model 491 prep cell is war ranted against defects in ma terials and workmanship for 1
year from date of purchase. If any defects oc cur in the in strument during this warranty period, Bio-Rad
Laboratories will re pair or replace the defective parts free of charge. The following defects, how ever, are
specifically ex cluded.
1. Damage caused by improper operation, accident, or misuse.
2. Repair or modification done by anyone other than Bio-Rad Laboratories.
3. Use of fittings or other parts supplied by anyone other than Bio-Rad Laboratories.
Note: This warranty does not apply to platinum wire electrodes.
For any inquiry or request for repair service, contact Bio-Rad Laboratories after confirming the model and
serial number of your instrument.
Table of Contents
Section 1 General Information .................................................................................1
1.1 Introduction ................................................................................................1
1.2 Accessory Equipment ................................................................................1
1.3 Specifications.............................................................................................2
1.4 Chemical Compatability .............................................................................2
1.5 Safety ........................................................................................................2
Section 2 Description of Major Components ..........................................................4
2.1 Model 491 Prep Cell Components .............................................................4
2.2 Lower and Upper Buffer Chamber .............................................................5
2.3 Cooling Core ..............................................................................................5
2.4 Gel Tube Assembly ....................................................................................6
2.5 Elution Chamber Base ...............................................................................6
2.6 Casting Stand ............................................................................................7
Section 3 Assembly and Operation .........................................................................8
3.1 Casting the Preparative Gel ........................................................................8
3.2 Preparing the Frits and Dialysis Membrane ...............................................10
3.3 Assembly of the Elution Chamber ............................................................10
3.4 Assembly of Upper and Lower Buffer Chambers ......................................12
3.5 Purge the Elution Chamber of Air Bubbles ...............................................14
3.6 Cooling the Gel ........................................................................................15
3.7 Loading the Sample .................................................................................16
3.8 Elution Rate, Detection, Collection, and Analysis ......................................16
Section 4 Optimizing Running Conditions for Preparative SDS-PAGE ................17
4.1 Gel Pore Size ...........................................................................................17
4.2 Optimization Procedures ..........................................................................18
4.3 Gel Length Determination for Preparative SDS-PAGE ...............................19
4.4 Gel Tube Size Selection ............................................................................19
4.5 Running Conditions ..................................................................................21
4.6 Elution Times for Proteins .........................................................................21
4.7 Examples of the SDS-PAGE Optimization Procedure ...............................22
Section 5 Disassembling and Cleaning .................................................................26
Section 6 Troubleshooting Guide ..........................................................................27
Section 7 Preparation of Electrophoresis Buffers and Acrylamide
Stock Solutions for SDS-PAGE .............................................................28
7.1 Separating (Resolving) Gel Buffer Stock ...................................................28
7.2 Stacking Gel Buffer Stock ........................................................................28
7.3 Sample Buffer (SDS-Reducing Buffer) Stock ............................................28
7.4 10x Electrode (Running) Buffer, pH 8.3 .....................................................29
7.5 30% Acrylamide Stock Solution ...............................................................29
Section 8 SDS-PAGE Gel Preparation ...................................................................30
8.1 Gel Recipes .............................................................................................30
8.2 Analytical Separating Gel..........................................................................31
8.3 Analytical Stacking Gel .............................................................................31
8.4 Preparative SDS-PAGE Separating and Stacking Gels ............................32
Section 9 A Guide to Preparative Native-PAGE ....................................................33
9.1 Introduction ..............................................................................................33
9.2 How to Choose Native-PAGE Systems ....................................................33
9.3 Discontinuous Native-PAGE ..................................................................... 36
9.4 Continuous Native-PAGE .........................................................................39
9.5 References ...............................................................................................42
Section 10 Ordering Information..............................................................................43
Section 1
General Information
1.1 Introduction
The Model 491 prep cell* is de signed to purify proteins or nucleic acids from complex mixtures by
contin uous-elu tion electrophoresis. Conventional gel elec trophoresis buffer sys tems and media are used
with the prep cell.
During a run, samples are electrophoresed through a cylindri cal gel. As molecules migrate through the gel
matrix, they separate into ring shaped bands. Individual bands migrate off the bottom of the gel where they
pass di rectly into the patented elution chamber for collection.
The elution chamber consists of a thin polyethylene frit. A dialysis membrane, directly underneath the elution
frit, traps proteins within the chamber. Elution buffer enters the chamber around the perimeter of a specially
designed gasket. The unique design of the gasket results in an even flow of buffer into the elution frit. Buffer
is drawn radially inward to an elution tube in the center of the cooling core. Purified molecules are drawn up
through the elution col lec tion tube at the cen ter of the cooling core by a peristaltic pump. The peristaltic
pump drives sepa rated pro teins through a UV monitor (optional) to a fraction col lector (Bio-Rad's Econo
system).
To assure that separated molecules migrate in compact, paral lel bands, temperature gradients across the
gel are minimized. The temperatures of the internal and exter nal surfaces of the gel are equalized by
continuously pumping lower electrophoresis buffer through the central cooling core by means of the buffer
recirculation pump.
™
Simple procedures are provided for determining optimal running conditions for most purifications. It is
recommended that these procedures be performed for each new sample to be purified before proceeding to
a preparative run with the Model 491 prep cell.
1.2 Accessory Equipment
Power supply — 500 volt
Warning: Use only power supplies with isolated ground such as Bio-Rad's PowerPac
PowerPac Universal
Buffer recirculation pump — (provided with the prep cell
Peristaltic pump (elution pump)*
Fraction collector*
UV monitor
Chart recorder**
* U.S. patent number 4,877,510
** The use of a reliable fraction collector is essential for the isolation of the de sired component of the sample. It may also be convenient
to monitor elution by UV ab sorbance. To simplify setup and operation of accessory equipment, we recom mend use of Econo system
low pressure chromatography components, in cluding peri staltic pump, fraction collector, UV monitor, and chart recorder.
™
HV or
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1.3 Specifications
Construction
Upper buffer chamber acrylic
Lower buffer chamber acrylic
Electrodes platinum, 0.010 inch diameter
Lid acrylic
Gel tube assembly glass/acrylic
Elution chamber base acrylic
Elution frit polyethylene
Support frit polyethylene
Cooling core glazed alumina
Elution tube borosilicate glass, 0.06” ID
Casting stand acrylic
Shipping weight 6 lb
Overall size 7 in. diameter x 14 in. high
Voltage limit 500 volts
Current limit 40 milliamperes
Power limit 20 watts
Cooling buffer flow rate 100 ml/min
Elution buffer flow rate 60 ml/hour
Upper electrophoresis buffer volume 300–600 ml
Elution buffer chamber volume 900 ml
Lower electrophoresis buffer volume 2–3 L
1.4 Chemical Compatibility
The Model 491 prep cell is not compatible with chlorinated hydrocarbons (e.g. chlo roform), aromatic
hydrocarbons (e.g. toluene, ben zene), or acetone. Their use will void all warranties.
1.5 Safety
Power to the Model 491 prep cell is to be supplied by an external DC power supply. This power
supply must be ground isolated in such a way that the DC voltage out put floats with respect to
ground. The recommended power sup ply for this instru ment is the PowerPac HV power supply.
The PowerPac universal power supply may also be used. The maximum speci fied operating
parameters for the Model 491 prep cell are:
500 VDC maximum operating voltage
40 mA maximum operating current
20 W maximum operating power limit
Current to the Model 491 prep cell, provided from the external power supply, enters the unit
through the lid assembly, providing a safety interlock to the user. Current flow to the cell is
broken when the lid assembly is removed. Do not attempt to cir cumvent this safety interlock,
and always turn the power supply off when working with the cell.
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The buffer recirculation pump is also ground isolated as should be any pump used with this cell. During
normal operation, the buffer in the lower buffer chamber is circulated through the cooling core and routed
back into the sys tem via the buffer recirculation pump. The buffer flowing through the tub ing and the pump
is electri cally active. For this reason handle the tubing carefully while the power supply is on. Do not touch
any exposed liquid when the power supply is on. Tube connections should be made with the power
supply turned off. Both the recir culation pump and the recommended power supplies are ground isolated by
design to minimize the potential shock hazard. However, working around high voltage equipment in a
laboratory environment is potentially dangerous. As a result it is the user’s responsibility to always excercise
care in setting up and running elec trophore sis instruments. If a liquid leak occurs, always turn off the power
supply be fore correcting the problem.
During operation, do not expose the cell to ambient temperatures above 50 ˚C.
Important
This Bio-Rad instrument is designed and certified to meet IEC1010-1* 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 manufacturer's warranty
• Void the IEC1010-1 safety certification
• Create a potential safety hazard
Bio-Rad is not responsible for any injury or damage caused by the use of this instrument for purposes other
than for which it is intended or by modifications of the instrument not performed by Bio-Rad or an authorized
agent.
*IEC1010-1 is an internationally accepted electrical safety standard for laboratory instruments.
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Section 2
Description of Major Components
2.1 Model 491 Prep Cell Components
Lid & power cables
Cooling buffer outlet
Elution buffer outlet
Upper buffer chamber
Cooling core
Elution buffer feedline
Gel assembly tube
Lower buffer chamber
Fig. 1. Exploded view of the Model 491 prep cell.
Elution chamber cap
Fluted gasket
Elution frit
Dialysis membrane
Support frit
O-ring
Elution chamber base
Cooling buffer inlet
Elution chamber
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2.2 Lower and Upper Buffer Chamber
The lower buffer chamber forms a stable base for the unit. It houses the an ode and contains the lower
electrophoresis buffer. The upper buffer chamber holds the up per electrophoresis buffer and the elu tion
buffer, and houses the cathode.
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Fig. 2. Upper and lower buffer chamber. Lower buffer chamber: Stopcock for lower elec trophoresis buffer inlet (1), and anode (2).
Upper buffer chamber: Elution buffer reservoir (3), upper electrophoresis buffer reservoir (4), ring nut (5), cooling buffer line (6), cooling
buffer feedline (7), and elution buffer feedline (8).
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2.3 Cooling Core
The cooling core extends to the bottom of the graduated gel tube and houses the elu tion tube in its center.
Cooling is recommended during polymeriza tion as well as during electrophoresis.
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Fig. 3. Cooling core. Cooling buffer ports (1), elution buffer outlet (2), cooling core place ment guides (3), and glazed ceramic cooling
core (4).
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2.4 Gel Tube Assembly
The gel tube assembly holds both the gel and the cooling core. The elution chamber cap and the gasket
mounted on the graduated gel column make up the upper part of the elution chamber. Two gel tube
assemblies are provided with the Model 491 prep cell: 28 mm ID and 37 mm ID. See Section 4.4 for
selecting the appropriate gel tube size for specific applications.
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Fig. 4. Gel tube assembly. Cooling core collar (1), upper reservoir attachment, i.e. threaded connector (2), graduated gel column (3),
elution chamber cap with thumb screws (4), elution buffer inlet port (5), cooling buffer port (6), gasket (7), and O-ring (8).
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2.5 Elution Chamber Base
The elution chamber base with the support frit holds the elution frit and the dialysis membrane directly
be neath the gel. The dialysis membrane provided with the Model 491 prep cell has a molecular weight cut
off of 6,000 daltons. A continu ous flow of elution buffer is directed through the channels of the elution
chamber gasket to the perime ter of the elution frit. As bands mi grate off the gel, they are washed to the
center of the frit, up through the elution tube in the center of the cooling core, and out to the peristaltic
pump, UV de tector (optional), and fraction collector. (The tracking dye, bromophenol blue (M
pass through the dialysis membrane. The flow of elution buffer through the elution frit overcomes the force of
elec trophore sis in the down ward direction.)
691), does not
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Fig. 5. Elution chamber base and tube assembly. Elution frit (1) and large O-ring (2).
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2.6 Casting Stand
Gels are cast with the gel tube assembly mounted directly on the casting stand. The casting stand ensures
that gels have perfectly flat lower surfaces. Inserting a spatula in the gel-release slot facilitates the removal of
the gel from the casting stand after polymerization by allowing air to enter beneath the gasket and the gel.
Fig. 6. Casting stand.
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Leveling feet (1), center pin (2), leveling bubble (3), and gel re lease slot (4).
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Section 3
Assembly and Operation
3.1 Casting the Preparative Gel
1. Place the gel tube assembly on the casting stand, aligning the four screws on the acrylic plate with the
holes in the casting stand. Secure the gel tube assembly with the four screws; hand tightening is
sufficient. Level the casting stand with the aid of the leveling bubble using the leveling legs.
2. Insert the cooling core so that the two placement guides slide through the grooves of the cooling core
collar in the gel tube assembly and the center pin on the casting stand is inserted in the elution tube of
the cooling core. Turn the core 90° until it locks into place. This will prevent any vertical or lateral
movement of the cooling core.
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3. It is advisable to cool the gel during polymerization. Cooling prevents ex cess heat accumulation in the
interior of the reaction mixture and aids in the formation of uniform gels. To cool, pump room temperature
water (or buffer) from an external source through the cooling core. Ensure that cooling is in progress prior
to casting the gel.
Fig. 7. Diagram of cooling path during polymerization. Cooling of gels during poly merization is recommended. This is accomplished
by circulating room temperature buffer between a reservoir and the cooling finger using the buffer recirculation pump.
4. Prepare the acrylamide monomer solution. Refer to Section 4 for selecting the appropriate acrylamide
gel concentration for a given application. Sections 8 and 9 describe preparative gel for mulations for
SDS-PAGE and native-PAGE, repsectively.
5. Pour the degassed monomer mixture into the gel tube through the gap be tween the cooling core and
the collar of the gel tube assembly. Avoid trap ping air bubbles in the gel. Gently tapping the casting
stand (with the gel tube assembly mounted to it) against the bench top will help to dislodge trapped air
bubbles. Visually inspect the gel for bubbles immediately after pouring the separating-gel solution into
the tube.
Carefully overlay the resolving gel with water-saturated 2-butanol or tert-amyl alco hol using the narrow
polytetrafluoroethylene (PTFE) tube affixed to a syringe (provided with the unit). Allow the resolving gel to
stand overnight for complete polymeriza tion (catalyst con centration in resolv ing gel is 0.025%
APS/0.025% TEMED). After 1–2 hours polymer ization, re place the alcohol overlay with gel buffer. In the
case of SDS-PAGE and Ornstein-Davis nondenaturing gels, this should be 0.375 M Tris/Cl, pH 8.8
buffer.
6. Very carefully decant or aspirate the buffer overlay. Cast the stacking gel, approx imately twice the
sample volume, on top of the resolving gel. Overlay the stacking gel monomer with water-saturated
2-butanol or tert-amyl alco hol. Allow the stack ing gel to polymerize for 1–2 hours.
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3.2 Preparing the Frits and Dialysis Membrane
Soak the elution manifold support frit, elution frit, and dialysis membrane in buffer. The frits must be completely wetted prior to use. To ensure removal of en trapped air in the pores of the frits, place the container
in which the frits are soaking in a vacuum chamber for approximately 10 minutes. Alternatively, the frits can
be soaked in buffer overnight to completely wet them. To maintain the wetting of the frits, store them in buffer.
The dialysis membrane provided with the Model 491 prep cell has a molecular weight cut off of 6,000 daltons.
Dialysis membranes with other pore sizes may be sub stituted for those pro vided. The dialysis membrane
must be soaked in buffer before use and stored in buffer or water between uses. If the membrane be comes
dry between runs, discard it. A properly stored dialysis membrane can be used at least for 5-6 runs. Prior
to each run, inspect the membrane care fully. Discard it if any holes or tears are detected.
3.3 Assembly of the Elution Chamber
1. Insert the soaked support frit into the base of the elution chamber. The stepped support frit is the
thicker of the two frits. Press the frit all the way into the base to form a flat surface on which to place
the dialysis membrane. Place the dialysis membrane on the support frit and the elution frit on the
dialysis membrane. Press the large elution O-ring in the groove around the perime ter of the base.
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2. Decant the stacking gel overlay, rinse the surface of the stacking gel with water, and loosen the four
screws holding the column to the casting stand. Carefully re move the gel tube assembly from the
casting stand. Insert a spat ula into the gel re lease slot and use it to gently pry the gel tube assembly off
the cast ing stand. Inspect the lower surface of the gel to make sure it is smooth. Trapped air bubbles
may cause a pitted gel surface which will result in un even elution of proteins from the gel. If a pitted or
otherwise uneven gel surface is observed, pour a new gel.
3. Place the gel tube assembly containing the gel on the elution chamber base. Align the four screws with
the holes in the elution chamber base and hand tighten them.
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