GE PhastSystem User manual

Phast System
user manual
automated electrophoresis
um 80-1320-15 Edition AI

3
Contents
1. Introduction .................................................................. 5
2. Important safety information ......................................... 9
2.1 Connection to the mains supply ............................... 9
2.2 Safety arrangements ............................................... 9
2.3 Safety precautions .................................................10
3. Description of the system ...............................................11
3.1 The separation and control unit ............................. 11
3.2 The development unit ...........................................15
3.3 PhastGel media and chemicals ...............................1
3.4 Using the keyboard .............................................. 22
3.5 The keyboard ......................................................23
4. Installation .................................................................31
4.1 Unpacking .......................................................... 31
4.2 Cable connections ................................................32
4.3 Turning the system on ..........................................33
4.4 Before use ........................................................... 33
5. Operation ...................................................................35
5.1 Programming separation procedures ...................... 35
5.2 Sample application ............................................... 39
5.3 Running IEF media .............................................. 41
5.4 Running electrophoresis media ..............................46
5.5 Programming development procedures ................... 50
5.6 Running a development method ........................... 54
5.7 Cleaning method .................................................57
5. Temperature compensation ................................... 59
6. Evaluation and presentation of data .............................65
6.1 Preservation ......................................................... 65
6.2 Evaluation ........................................................... 67
. Maintenance and trouble shooting ................................ 3
7.1 Separation and control unit ................................... 74
7.2 Development unit ................................................. 76
7.3 Trouble shooting ..................................................7
8. Ordering information and technical data....................... 9
.1 Ordering information ...........................................79
.2 Technical data ..................................................... 1
1. Contents

4
Important user information
Reading this entire manual is
recommended for full
understanding of the use of
this product.
The exclamation mark within an equilateral triangle
is intended to alert the user to the presence of
important operating and maintenance instructions
in the literature accompanying the instrument.
The lightning symbol within an equilateral
triangle is intended to alert the user to the
risk of exposure to high voltages.
The earth (ground) terminal symbol is used
to mark a functional earth terminal.
Should you have any comments on this manual, we
will be pleased to receive them at:
GE Healthcare Bio-Sciences AB
S-751 2 Uppsala
Sweden
GE Healthcare Bio-Sciences AB reserves the right to
make changes in the specifications without prior
notice.
Warranty and Liability
GE Healthcare Bio-Sciences AB guarantees that the
product delivered has been thoroughly tested to
ensure that it meets its published specifications. The
warranty included in the conditions of delivery is
valid only if the product has been installed and used
according to the instructions supplied by
GE Healthcare Bio-Sciences AB.
GE Healthcare Bio-Sciences AB shall in no event be liable
for incidental or consequential damages, including
without limitation, lost profits, loss of income, loss of
business opportunities, loss of use and other related
exposures, however caused, arising from the faulty
and incorrect use of the product.
Trade marks
PhastSystem™, PhastTransfer™ and PhastGel®
are the exclusive trade marks of
GE Healthcare Bio-Sciences AB.
In view of the risk of trade mark
degeneration, it is respectfully suggested that
authors wishing to use these designations refer to
their trade mark status at least once in each article.
Copyright© 1995 GE Healthcare Bio-Sciences AB
All rights reserved. No part of this product may be
reproduced, stored in a retrieval system or transmit-
ted in any form by any means, without permission in
written form from the company.

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1. Introduction
PhastSystem consists of a separation and control unit, a development
unit, high-performance PhastGel® separation media, accessories, and a
technical support package. These components work together to form
a system for fast, high-resolution, and reproducible electrophoresis.
With PhastSystem, isoelectric focusing is as easy to perform as gel
electrophoresis; Coomassie staining is as easy as silver staining. The
schematic diagram below illustrates the steps involved in producing a
finished electrophoresis gel using PhastSystem with PhastGel
separation media.
Flow diagram or PhastSystem
™
▼
▼
▼
▼
▼
▼
Separation and control unit Development unit
Place 1 or 2 gels on Place gel(s) in
the separation bed 3 min. development chamber 1 min.
Load PhastGel Select a programmed
sample applicator(s) 3 min. development method
and press the start button
Select a programmed when method stops 3 -9 min
separation method
and press the start
button
remove the gel(s) and
analyze the results
when alarm sounds 2 -45 min. Total time 32-92 min.
remove the gel(s)
Total time 26-5 min.
or
Specific detection via
conventional methods
e.g. zymograms, auto-
radiography, blotting
The time intervals listed above will depend on the technique that is run.
▼
▼
▼
1. Introduction

6
This users guide includes the following chapters:
Chapter 2: Important safety information.
Chapter 3: Description of the system; introduces you to
PhastSystem.
Chapter 4: Installation; tells you how to install PhastSystem.
Chapter 5: Using the keyboard; prepares you for programming
and running methods.
Separation procedures, shows you how to program
and run separation methods.
Development procedures, shows you how to
program and run development methods.
Chapter 6: Evaluation and presentation of data; gives advice
on drying, mounting, and photographing gels and
describes procedures for molecular weight and
isoelectric point measurement using calibration
proteins.
Chapter 7: Maintenance and trouble shooting; shows you
how to replace and clean certain parts of the
instruments and how to calibrate the temperature
sensors. Provides current trouble shooting
recommendations. If you have any problems
during programming or operation you find all help
messages listed here.
Chapter : Ordering information and technical data; gives you
all information needed to order the products
mentioned in this manual. You will also find a list
of the most common spare parts required for
maintenance of PhastSystem.
A list of the technical data on PhastSystem
instruments and PhastGel media and accessories is
also included.
Chapter 9: Separation technique files; you will find optimized
methods for a number of separation techniques.
Development technique files; you will find
optimized methods for a number of development
techniques.
Application notes; here you can file application and
technical notes covering specific techniques or
application areas.
A technological extension of PhastSystem is PhastTransferTM.
PhastTrans er
PhastTransfer brings speed, reproducibility and convenience to semi-
dry electrophoretic transfer of proteins from PhastGel separation
media to immobilizing membranes. The small format of the gels
together with semi-dry transfer method minimize the amount of
reagents needed for detection. Elution efficiency is greater than 90%
for most protein systems. At 1.0 mA/cm2, high transfer recovery is
obtained, usually within 10–30 minutes.
1. Introduction

7
Phastsystem
Fig.1. PhastSystem consists of a separation and control unit, a development unit,
PhastGel separation media, accessories and a technical support package.
1. Introduction

9
2. Important safety
information
Voltage selector setting
The instruments are available in two versions: one for 220-230/240
V AC, referred to here as the 220 V model, and one for 100/120 V
AC, referred to here as the 120 V model.
As a safety precaution, check the code number and voltage printed
on the backpanels to ensure you have the correct model for your
local electricity supply.
Code number 1 -101 -23:
Separation and control unit 120 V model
Development unit 120 V model
Code number 1 -101 -24:
Separation and control unit 220 V model
Development unit 220 V model
Set the voltage selectors on the rear panels of the separation and
control unit and development unit according to your local electricity
supply. To do this:
• Check the voltage range of the mains electricity supply.
• Set the voltage selector to the appropriate setting according to
the table below.
Voltage range Voltage selector setting
For12 V model instruments:
9 -11 1
1 8-132 12
For 22 V model instruments:
198-242 22 -23
216-264 24
Important! Always disconnect the mains power cords when
servicing the system.
The operator is protected against high voltage by the separation
compartment lid when an electrophoresis is in progress.
If the lid is opened during a run, the high voltage supply switches off
automatically to eliminate electrical hazard. An alarm will sound
until the lid is closed or until the run is paused.
2.1 Connection to
the mains supply
2.2 Sa ety
arrangements
2. Important safety information

10
The voltage supplied by PhastSystem is capable of delivering a lethal
electric shock. The numerous safety devices and circuits built into the
instrument prevent this. The “pause” and “start/stop” keys can also
be pressed to halt the supply of power at any stage of the experiment
or operation of PhastSystem. Nevertheless, in keeping with good
laboratory practise, we advice you to take the following precautions
when dealing with the instrument.
1. Regularly check all insulation cables, take care not to damage
the units, especially the separation compartments lid.
Note: For full safety it is important that the lid is not
tampered with.
2. Ensure that the mains cables are plugged into fully grounded
mains outlets.
3. Allow only authorized service representatives to service or work
on the electrical circuitry of PhastSystem.
4. Avoid spilling buffers or other conduction liquids onto the
instrument.
5. Allow the ventilation slots (situated at the rear of PhastSystem)
to have free access to a good flow of air.
2.3 Sa ety
precautions
2. Important safety information

11
3. Description of the
system
The aim of this chapter is to introduce you to PhastSystem. Each
component of PhastSystem is described in turn; the separation and
control unit, the development unit, and PhastGel media and
chemicals. After you have read this chapter you will know what the
components look like, how they function, and how they work
together to form a system for fast electrophoresis, PhastSystem.
The separation and control unit is the heart of PhastSystem because it
contains the microprocessor which controls and monitors both
separation and development processes according to programmed
methods. Methods are programmed using the keyboard. The LCD
display shows the method steps during programming. When
separation and development methods are started, the display shows
the actual running conditions so you can monitor the progress of the
methods.
The separation and control unit also contains the separation
compartment and the power supply. The microprocessor, separation
compartment and power supply are described below. The keyboard is
described in a following chapter.
Microprocessor
The microprocessor in the separation and control unit controls and
regulates all parameters during separation and development runs.
Methods are programmed using the keyboard, and stored in a
semiconductor memory. This memory is guarded by a battery so that
methods are not lost when the system is turned off or if mains power
fails.
Every time the system is turned on, the microprocessor does a
diagnostic test to make sure everything functions properly. If an error
is detected a message will appear on the display.
The microprocessor will also detect programming errors or instru-
ment malfunctions during operation. In this case, an alarm will
sound, running methods will be paused, and a message will appear
on the display telling you what is wrong. These messages, called help
messages, are listed by number in chapter 7, where you can find more
information about trouble shooting.
Separation compartment
The separation compartment in the separation and control unit
contains a separation bed with positions for two gels. There are two
alternate positions for each gel. The vertical position, with the tab at
the front, is the normal position. The horizontal position, with the tab
to the left, is for running the second dimension in electrophoretic
titration curves.
3.1 The separation
and control unit
3. Description of the system

12
The Peltier element automatically cools and heats the separation bed
to the programmed temperature. The programmable temperature
range extends from 0°C to 70°C (see cooling capacity, page 14). The
heat generated during electrophoresis is transferred to a large air
cooled heat sink.
A standby temperature can be programmed to cool (or heat) the
separation bed before methods are started. This saves time since a
method will not start until the bed temperature equals the
programmed temperature for the first step in that method.
The electrode assembly contains two anodes (+), and one cathode (-)
for each gel. The electrodes are made of platinized titanium. An
assembly with reversed polarity is also available for electrophoresis of
basic proteins in their native state. A high voltage power supply, inside
the separation and control unit, generates the required electric field
for electrophoresis (see power supply, page 13). If the lid is opened
during a run, the high voltage supply switches off automatically to
eliminate electrical hazard. An alarm will sound until the lid is closed
or until the run is paused.
Fig. 2. Separation compartment
Sample application
Samples are applied to gels with PhastGel sample applicators. These
small, comb-like pieces have a series of capillary wells. Samples are
drawn into the capillaries and held there until the applicator is low-
ered onto the gel at a set time in the program. Once the applicators
are loaded with samples they are placed into one of the slots in the
sample applicator arm.
The sample applicator arm has four alternative sample applicator
positions for each gel. The position nearest the cathode is for PhastGel
electrophoresis media, and the other three positions are for PhastGel
IEF media. The plunger toward the back of the compartment holds
the applicator arm up until it is time for sample application.
The electrode assembly and the applicator arm are raised so the gels
can be positioned onto the separation bed. When lowered again, the
3. Description of the system

13
electrode assembly may take up two horizontal positions, depending on
the setting of the two eccentric levers. The lower position is used for
PhastGel IEF media, where the inner electrodes (the anode nearest the
cathode and the cathode) rest directly on the gel. The higher posi-
tion is used for PhastGel electrophoresis media, where the outer elec-
trodes rest on PhastGel buffer strips which are held in place on the gel
by the PhastGel buffer strip holder.
The buffer strip holder, like the IEF gel cover, also serves to prevent gels
from drying out during electrophoresis.
Fig. 3. Sample application.
Separation methods
Nine separation methods are available for programming. For each
method, you can program two sample application instructions (for
lowering and raising the sample applicators), an extra alarm
instruction, and up to nine steps. For each step, the voltage, current,
power, separation bed temperature, and duration of the step in
volthours is programmed.
Before a separation method is started, the sample applicator arm rests
a few millimeters above the gels. After a programmed interval during
the run, the applicator arm is lowered to apply the samples to the
gels. After a programmed interval, the applicator arm is raised again.
An alarm will sound to mark the end of the last step in a running
method. But, methods will continue to run with the same running
conditions as the last step until the method is stopped by pressing the
stop key. This is to prevent band diffusion in case you miss the alarm.
Power supply
The power supply can be programmed to function in three modes:
constant current, constant voltage, or constant power, by setting
limits on these parameters. The microprocessor automatically adjusts
the parameters during each step in a separation method.
3. Description of thae system

14
For maximum reproducibility, the duration of each method step and the
time for sample application is measured in volthours. Volthours indicate
the extent of protein migration in the gel since electrophoretic mobility is
proportional to the applied voltage and the time that this voltage is
applied. Since the voltage change continually, the unit is equipped with
a volthour integrator, which integrates volts with time. The extra alarm
is also programmed in volthours. For more information about
volthours and volthour integration, see reference 1.
1. Isoelectric Focusing. In Gel Electrophoresis and Isoelectric
focusing of Proteins, Allen, R.C., Saravis, C.A., Maurer, H.R.
(editors), Walter de Gruyter, Berlin and New York, 19 4, p. 76,
Allen R.C.
Cooling capacity
The cooling capacity of the separation bed will depend on the
following: 1) the ambient temperature; 2) the power applied to the
gels; and 3) if one or two gels are run. Fig. 4 below illustrates the
separation bed temperature versus time for native PAGE, SDS-PAGE,
and IEF runs. The running conditions are given in the caption under
the graph. A slight temperature drift can be seen for the IEF run with
an ambient temperature of 2 °C. Even with an ambient temperature
of 3 °C, no temperature drift is experienced with native gradient and
SDS-PAGE runs.
Fig. 4. Separation bed temperature vs time. The plots represent the following
conditions: l) IEF (PhastGel IEF 3-9); 2 V 5 mA, 7 W 1 °C, with 23°C ambient
temperature; 2) Same as 1) but with an ambient temperature of 28°C; and 3) Native
gradient SDS-PAGE (PhastGel gradient media); 4 V 15 mA, 4W 15°C; with ambient
temperature of 38°C.
3. Description of the system

15
Fig. 5 below shows the lowest separation bed temperature maintained
(within ±l°C) for separations run at 4 and 7 watts with different
ambient temperatures. Lower temperatures can be achieved but
temperature drifts exceeding ±l°C might occur. Depending on the
magnitude of the temperature drift, results may or may not be
affected. Therefore, use this graph as a guide, not as a rule. When
choosing a separation bed temperature, the humidity in the room
must also be taken into account, or excessive condensation might
affect results.
Fig. 5. Cooling capacity vs ambient temperature.
The lowest separation bed temperature achievable (with deviations less than ±1°C)
with ambient temperature up to 4 °C for two gels run at 4 W and 7 W.
The visible parts of the development unit are: a stainless steel chamber
(with a heating foil), a rotating gel holder for one or two gels, a
temperature and level sensor on the underside of the lid, and ten ports
through which the development chamber can be filled and emptied.
Ports labelled 1-9 are used to connect development solutions to the
development chamber. The port labelled 0 is reserved for waste, that
is, solutions only exit through this port. The gel holder, liquid level
sensor, and temperature sensor are mounted in the lid of the
development chamber and protrude into the chamber when the lid is
closed.
Inside the unit there is a pneumatic pump for filling and emptying the
chamber, a 10-port valve for the selection of ports, and a 3-port valve
for the selection of pump functions i.e., creating vacuum or pressure
in the chamber.
The pneumatic pump is connected to an opening in the lid of the
chamber. A gasket in the lid makes the chamber airtight when the lid
is closed. By creating a vacuum in the chamber, liquid is drawn in
3.2 The
development
unit
3. Description of the system

16
through a hole in the bottom of the chamber. Similarly, by creating
excess pressure in the chamber, liquid is pushed out through the same
hole in the bottom.
Development methods
Nine development methods are available for programming. For each
method, you can program up to 20 steps. For each step, the in-port
for filling, the out-port for emptying, the duration of the step in
minutes, and the temperature for processing the gel (the chamber can
heat solutions up to 50°C) is programmed.
As programming options, each development method can have a
temperature compensation curve, and an extra alarm (to sound at a
set time during the run). More information about temperature
compensation is given in Development procedures, section 5.3.
Once the bottles of development solution are connected to the ports
(by the PVC tubing), the gels are inserted into the gel holder, the lid is
closed, the start button is pressed, and the rest is automatic. The
method ends when it reaches an empty (unprogrammed) step.
Fig. 6. Development chamber.
3. Description of the system

17
Fig. 7. 1 -port valve.
Chemical resistance
The parts that come into contact with development solutions in the
development unit are resistant to chemicals typically used in
Coomassie and silver staining, for example acetic acid, methanol, and
silver staining solutions. If you plan to use other chemicals, for
example, to clean the unit, you should first check the resistance of the
wetted parts to the chemical in question.
The chemical resistance of a polymer depends on many factors,
including the temperature and concentration of the solution, the
application (a compound that swells may function well as a static seal,
yet fail in dynamic applications), and the period of exposure. Table 1
below is intended as a general guide for the chemical resistance of the
wetted parts in the development unit.
If you are in doubt about the resistance of wetted parts to a certain
chemical, test the parts first; order spare parts for such tests (see
Ordering information, chapter ).
In general you should avoid using ketones, hot strong acids, and
organic hydrocarbons.
3. Description of the system

1
Table 1: A general guide for the chemical resistance of the wetted parts in the
development unit.
Wetted parts1Material o Generally Generally
construction resistant to attacked by
Distributor and PVDF2strong acids and ketones, esters, and hot
distributing plate PVDF2bases in moderate acids
concentration and
alcohols and
hydrocarbons
Gasket (1 -port fluoro rubber moderate acids, hot strong acids, esters,
valve) strong bases, ketones, and bleach
many solvents,
alcohols,
aldehydes
Tubin (1 -port Teflon most chemicals extreme conditions
to chamber)
Tubing (bottles PVC3strong acids and hot acids, ketones, and
to 1 -port valve) bases in moderate hydrocarbons
concentration,
alcohols,
aldehydes, and
bleach
Chamber, gel stainless most chemicals long exposure to salt
holder, and temp. steel solutions
sensor
Chamber lid gasket EPDM4strong acids and hot acids and aromatic
bases, alcohols, hydrocarbons
aldehydes, and
ketones
Chamber lid PP5strong acids in hot strong acids, aromatic
moderate con- hydrocarbons, and bleach
centration in high
concentration,
alcohols, alde-
hydes, and
ketones
1These parts are illustrated on pages 16, 17 and 79. 4Ethylene propylene copolymer and terpolymer
2Polyvinylidine fluoride 5Polypropylene (or polypropene)
3Polyvinyl chloride
At present, PhastGel media are available for four types of electrco-
phoretic techniques; native polyacrylamide gel electrophoresis
(PAGE) in gradient or homogeneous gels, SDS-PAGE in gradient or
homogeneous gels, and isoelectric focusing (IEF). These gels can be
combined for twodimensional techniques.
PhastGel media are made of polyacrylamide bonded to a transparent
polyester backing. The gel surface is covered with a plastic film which
prevents drying and contamination. This film must he peeled off
directly before use, that is, after the gel has been positioned onto the
separation bed, and excess water has been removed.
PhastGel media are individually packaged in airtight envelopes. Once
removed from their package, gels should be used immediately.
PhastGel chemicals include buffer strips for electrophoresis and a
Coomassie-type stain, PhastGel Blue R. PhastGel media and chemi-
3.3 PhastGel media
and chemicals
3. Description of the system

19
cals are described below. See Separation procedures and Development
procedures for detailed instructions for using these products.
PhastGel IEF media
PhastGel IEF media are homogeneous (5% T, 3% C) polyacrylamide
gels containing Pharmalyte® carrier ampholytes. Pharmalyte
generates stable, linear pH gradients with a smooth conductivity
profile across the entire pH range, which means that high field
strengths of 500 volts/cm and above can be used. Three different
PhastGel IEF media are available: PhastGel IEF 3-9, 4-6.5 and 5- .
PhastGel IEF media are run without buffer strips.
The histogram shown here illustrates the pH ranges of PhastGel IEF
media with respect to the pI distribution of 00 proteins. (See
Technical data, chapter , and Separation technique file No. 100,
chapter 9, for further details.)
PhastGel electrophoresis media
PhastGel electrophoresis media are used together with PhastGel
buffer strips. Buffer strips, made of high quality agarose with low
electroendosmosis and high purity reagents, serve as buffer reservoirs
to generate discontinuous buffer systems in the gels during a run.
During a separation, proteins are first concentrated in a porous
stacking gel zone, they then move into the separation gel zone where
they are separated according to size. The migration distance of a
protein is related to the logarithm of its molecular weight (MW).
Molecular weights are easily estimated using one of the GE Healthcare
molecular weight calibration kits. (See Evaluation and
presentation of data, chapter 6, for instructions.)
Seven different gels for electrophoresis are available, three for gradient
gel electrophoresis and four for homogeneous gel electrophoresis. The
three gradient gels are PhastGel gradient 10-15 with a continuous
gradient from 10 to 15% polyacrylamide, PhastGel gradient -25
with a continuous gradient from to 25%, polyacrylamide and
PhastGel gradient 4-15 with a continuous gradient from 5-15% total
polyacrylamide and a 1-2% gradient cross linker. Three of the homo-
geneous gels are PhastGel homogeneous 7.5, PhastGel homogeneous
12.5 and PhastGel homogeneous 20, with a concentration of 7.5,
12.5 and 20% polyacrylamide respectively. The fourth homogeneous
gel is PhastGel high density which has a polyacrylamide concentration
of 20% and a 30% concentration of ethylene glycol. (See Technical
data, chapter , and Separation technique files 111, 112, 120, 121
and 130, chapter 9, for further discussion and details.)
3. Description of the system

20
Fig. 8. The approximate pH ranges of PhastGel IEF media are superimposed on a
histogram showing the isoelectric point. The histogram is made up of data from 8
proteins. (Gianazza, E., Righetti, P.G., J. Chromatography 193 (198 ) 1-8.) By kind
permission of the authors and publisher.
The two histograms shown here illustrate the molecular weight ranges for
PhastGel gradient media with respect to the molecular weight
distribution of proteins in both denatured and non-denatured form.
Fig. 9. The approximate molecular weight separation ranges of PhastGel gradient
media are superimposed on a histogram showing the molecular weight distribution of
denatured proteins. The histogram is made up to data collected from 53 proteins.
Each bar spans 1 , daltons. (Gianzza, E., Righetti, P.G., J. Chromatography, 193
(198 ) 1-8). By kind permission of the authors and publisher.
3. Description of the system