Hoefer SE 400 User manual

user manual
SE 400/SE 410
um SE400-IM/Rev. A0/06-04
Hoefer SE 400/SE 410
the Sturdier vertical slab gel electrophoresis units

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Page finder
1. Gel electrophoresis unit function and description
Unpacking and disassembly ............................................ 2
Annotated inventory ........................................................ 4
Specifications ................................................................ 6
2. Operating instructions
2.1. Gel casting preparation
2.1.1. options—precast and self cast gels ............ 7
2.1.2. preliminary casting steps ........................... 8
2.2. Acrylamide gel preparation
2.2.1. resolving gel ........................................... 11
2.2.2. stacking gel ............................................ 12
2.2.3. gradient gel ............................................ 13
2.3. Sample preparation ........................................... 15
2.4. Final assembly ................................................. 16
2.5. Resolving the sample ........................................ 20
2.6. After electrophoresis ......................................... 23
3. Care and maintenance ........................................... 24
4. Troubleshooting ...................................................... 25
Appendices
A. Laemmli System Gels ............................................... 29
B. Bibliography ............................................................ 37
Ordering information ................................................... 39

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English
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Safety warnings and precautions
• Only use a power supply that is CE marked or
safety certified by a nationally recognized testing
laboratory.
• Turn all power supply controls off before plugging
in or removing the power leads, and only connect or
disconnect leads when the safety lid is in place.
• Do not operate at temperatures above 45 °C.
All plastic parts are rated for 45 °C continuous
duty. Overheating will cause irreparable damage to
the unit!
• If this equipment is used in a manner not specified
by the manufacturer, the protection provided by the
equipment may be impaired.
• Only accessories and parts approved or supplied by
Hoefer, Inc. may be used for operating, maintaining,
and servicing this product.
• Utiliser seulement un générateur marqué CE ou
dont la sûreté a été certifiée par un laboratoire de
test nationalement reconnu.
• Eteindre le générateur avant de brancher ou de
débrancher les prises. Le couvercle de sécurité doit
être en place avant de brancher ou de débrancher
les prises au générateur.
• Ne pas utiliser à une température au dessus de
45 °C. Toutes les pièces en plastique sont prévues
pour résister à une température constante de 45 °C.
Toute surchauffe causera des dommages irrépara-
bles à l’unité.
• Si cet appareil est utilisé de manière incorrecte ou
non approuvée par le constructeur, la protection
fournie par l’appareil peut etre altérée.
• Seuls les accessoires et pièces détachées approuvés
ou fournis par Hoefer, Inc. sont recommandés pour
l’utilisation, l’entretien et la réparation de
ce produit.
Français
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1. Unit function and description
The Hoefer™ SE 400 and SE 410 Sturdier™ Verti-
cal Slab Gel Electrophoresis Units are intended
for electrophoretic separation of proteins and
nucleic acids under both denaturing and native
conditions. Up to 28 samples can be compared
on a single slab gel. One gel (or two gels if using
the divider plate, ordered separately) is cast in
the casting stand side of the unit. The size of
the gel is 14 × 15 cm if using the SE 400, and
14 × 23 cm if using the SE 410. After casting,
the sandwich is transferred into the lower buffer
chamber for electrophoresis.
The basic unit includes one set of glass plates
(18 × 16 cm for the SE 400, and 18 × 24 cm for
the SE 410), two clamp assemblies (SE 400: two
16 cm clamps; SE 410: two 16 cm clamps and
two 8 cm clamps), and two cams. The complete
unit includes one 15-well comb and two spacers,
1.5 mm thick, in addition to the basic unit.

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Unpacking and disassembly
Unwrap all packages carefully and compare
contents with the packing list, making sure all
items arrived. If any part is missing, contact
your local sales office. Inspect all components
for damage that may have occurred while the
unit was in transit. If any part appears damaged,
contact the carrier immediately. Be sure to keep
all packing material for damage claims or to use
should it become necessary to return the unit.
This unit is partially assembled to protect
components during shipping. To disassemble:
1
Position the unit so that the electrical connectors
face you.
2
Note the holes at each side on the upper buffer
chamber. Rest your thumbs in these holes and use
your index fingers to lift the sides of the safety lid
gently until the electrode connectors unplug. First lift
the lid straight up so that the upper electrode shield
clears the upper chamber and then lift the lid out
(toward you) to remove it completely.
3
Lift out the upper buffer chamber and then the glass
plate assembly.
4
Remove the clamps by loosening the thumb screws.

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color-coded connectors (2)
color-coded leads (2)
safety lid
upper buffer chamber
glass plates (2)
casting cradle
leveling feet (4)
clamps
lower buffer chamber
Fig 1. SE 400 series
main components.
Included but not shown:
Cams
GelSeal grease, 1/4 oz.
Spacer-Mate
Wonder Wedge
Level
Required but not included:
Approved power supply

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Annotated inventory
Buffer chambers. Both buffer chambers are
chemically resistant to common electrophoretic
buffers but not to organic solvents or strong
acids and alkalis.
Safety lid. The lid contains both electrodes and
both electrode connectors. The electrode connec-
tors plug into the lead connectors on the lower
buffer chamber. The color-coded leads plug into
color-coded jacks on the power supply.
Glass plates. Two 18-cm wide glass plates are
included. Plates for the SE 400 are 16 cm long,
and plates for the SE 410 are 24 cm long.
(A notched divider plate, ordered separately,
can be used to run two gels at the same time.)
Clamps. Two 16-cm clamps are required to
secure the 16 cm long sandwich. These and an
additional pair of 8-cm clamps are required to
secure a 24-cm long sandwich.
Casting stand. The caster can be leveled with
the adjustable leveling feet on the bottom of the
unit. A laminated gasket seals the bottom of
the glass plate assembly when it is locked into
the stand.
Cams. Cams are used twice; first to secure the
assembled sandwich in the casting stand and
again to lock the sandwich and the upper buffer
chamber together.
Rubber gaskets. There are two gaskets. The
laminated gasket fits into the bottom of the cast-

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ing stand and provides the seal for the bottom
of the gel sandwich. The slotted gasket fits
under the upper chamber and provides the seal
between the sandwich and the upper chamber.
Two ridges help position this gasket.
Spacer-Mate assembly template. Aligns spacers
for sandwich assembly.
Wonder Wedge Plate Separator Tool. Use to
disassemble gel sandwiches and to gauge spacer
and comb thickness.
Spacers. (May be ordered separately.) Spacers
determine the thickness of the gel. They are 2 cm
wide and are available in three thicknesses: 0.75,
1.0, and 1.5 mm.
Combs. (May be ordered separately.) Teflon™
combs that form 1 to 28 wells are available in
three thicknesses: 0.75, 1.0, and 1.5 mm. Blank
combs form a single large well, and preparative
combs include 1 or 2 reference wells in addition
to the preparative well.
All blanks, preparative combs, and combs with
fewer than 28 wells form wells that are 25 mm
deep. The 28-well comb forms wells that are
only 15 mm deep so that wells do not collapse
when the comb is removed. The sample volume
held by each well depends on the gel thickness,
well depth and the number of wells per comb.
Table 2 on page 15 lists volume per 1 mm depth
for wells created by each comb size. See the
ordering information for additional comb
specifications.

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Specifications
Glass plate size SE 400: 18 × 16 cm
SE 410: 18 × 24 cm
Approximate gel size SE 400: 14 × 15 cm
SE 410: 14 × 23 cm
Max. wattage 20 W
Max. voltage 500 V @ 40 mA
Max. amperage 30 mA/gel
(60 mA total @ 325 V)
Max. temperature 45 °C
Environmental Indoor use: 4–40 °C
operating conditions Humidity up to 80%
Altitude up to 2000 m
Installation category: II
Pollution degree: II
Dimensions (w × h × d) SE 400: 24 × 28 × 15 cm
(9.5 × 11 × 6 in.)
SE 410: 24 × 36 × 15 cm
(9.5 × 14.2 × 6 in.)
Product certifications EN61010–1, UL3101–1,
CSA, C22.2 1010.1, CE
This declaration of conformity
is only valid for the instrument
when it is:
• used in laboratory locations,
• used as delivered from
Hoefer, Inc. except for
alterations described in
the User Manual, and
• connected to other CE
labeled instruments or
products recommended or
approved by Hoefer, Inc

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2. Operating instructions
Procedures for casting gels and electrophoretic
separation follow. Included are instructions for
both single percentage (homogeneous) and
gradient polyacrylamide gels. Appendix A lists
recipes and Appendix B gives a bibliography.
2.1. Gel casting preparation
2.1.1. options—precast gels and self-cast gels
The SE 400 unit accepts standard precast gels
purchased from commercial suppliers as well
as self-cast gels, which can be prepared using
the built-in casting stand. (To cast multiple
14 × 16 cm gels, the Multiple Gel Caster Kit,
which holds up to 10 sandwiches, and the Gel
Caster Kit, which holds up to four sandwiches,
can be ordered separately.) Gels for the SE 410
must be self-cast.
Glass plates, spacers, and clamp sets are sized
so that the assembled sandwich can be easily
aligned to create the required seal. When assem-
bling sandwiches, take extra care to align all
components for best results.

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Fig 2. A 24-cm sandwich requires
two 16-cm and two 8-cm clamps.
24 cm
(SE 410)
16 cm
(SE 400)
2.1.2. preliminary casting steps
1
Prepare the caster
Place the spirit level into the lower buffer chamber
and adjust the leveling feet.
2
Prepare the clamps
Loosen all clamp screws and make space for the
sandwich by sliding the pressure plates toward
the screws.
3
Construct each gel sandwich
For each sandwich, choose two perfectly clean
unchipped glass plates and two spacers. Lay one
plate on a flat surface, lay the Spacer-Mate assembly
template onto the plate (wide side at the top), place
a spacer along each edge, and lay the second glass
plate on top.
4
Secure the sandwich with clamps
Slide one clamp at a time along the sandwich sides.
Finger tighten one screw on each clamp, set the
sandwich upright on a flat surface, and loosen the
screw to align the stack. Take great care in aligning to
ensure a seal. Finger tighten all screws. Remove the
Spacer-Mate.
24-cm sandwich (SE 410)
A 24-cm sandwich requires two clamp assemblies on
each side. Align each end separately. That is, align
one end, finger-tighten the screws, turn the sandwich
180° and align the other end. In each case allow the
clamp to slide down and align perfectly with the top
(or bottom) edge of the glass plates.
For 24 cm long plates, position the 8 cm clamp at
the bottom (see Fig 2).

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2-gel sandwich
A 16- or 24-cm long notched divider plate (ordered
separately) doubles the number of gels that can be
cast and run (see Fig 4).
Assemble in the same manner as a single gel sand-
wich, except before placing the top glass plate, lay
the divider plate atop the first set of spacers and a
second set of spacers atop the divider plate. Place the
notch so that it will be at the top of the gels. As with
a regular sandwich, it is essential that the spacers and
plates align perfectly in order to create a seal.
5�
Inspect the bottom of the sandwich to make sure that
edges are aligned flush in order to ensure a complete
seal. Adjust if necessary (see Fig 3).
Optional: Apply a light film of GelSeal only on the
bottom outside corners if your sandwiches tend to
leak. Do not use silicone grease or petroleum jelly
to seal the sandwich because these substances are
difficult to remove and ultimately may cause artifacts
in the gel.
6��
Place the laminated gasket into the casting cradle
with the foam side down. Place the glass plate
assembly in the casting cradle, screw side facing
out (see Fig 5).
24-cm plates: Place the sandwich so that the short
clamps are at the bottom.
7�
Insert a cam into the hole on each side of the casting
tray with the ridge (short end) pointing up. Seal the
gel sandwich by turning both cams as far as needed,
usually 90° to 150°, up to 180°. The cam action
presses the plates into the gasket to seal the bottom
of the sandwich. The seal is complete once the glass
edge appears darker and nearly transparent against
the gasket. Do not tighten the cam past this point.
Fig 4. 2-gel sandwich assembly:
2-gel sandwiches are limited to
thinner gels; no spacers thicker
than 1.5 mm can be used.
glass plates
(at the outer sides
of the sandwich)
spacers
notched
divider
plate
Tip: Remove the laminated
gasket from the cradle and use
the casting cradle to hold the
sandwich for alignment.
Fig 3. Sandwich assembly:
Inspect glass plates for nicks.
Use only unchipped plates to
prevent leaking.
pressure bar
both top and bottom
sandwich edges must
be flush with the
clamp guide ridges.

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Note: It is easier to keep the caster balanced if you
turn both cams toward the center of the caster.
glass plates (2)
clamps
(the number required
depends on the
plate length.)
spacers
leveling feet (4)
casting cradle
cams (2)
insert the cam(s) in
the cam holes and
turn toward the center
to lock the glass plate
assembly.
gasket (foam side down)
Fig 5. Caster components
and assembly.
1. Lower the assembled sandwich
into the casting cradle.
2. Insert cams into the cam holes,
ridge end up.
3. Turn cam up to 180° until
the glass plates seal against
the gasket.

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2.2. Acrylamide gel preparation
Table 1. Approximate monomer solution volume
required for a single gel
Gel thickness (mm)
Model 0.75 1.00 1.5
SE 400 15 ml 23 ml 30 ml
SE 410 23 ml 34 ml 45 ml
2.2.1. resolving gel
1��
Prepare the monomer solution and pour the gel.
Prepare the required amount of monomer solution,
deaerate, and add the initiator and catalyst just prior
to pouring the gel.
2��
Pipet the solution into one corner of the sandwich,
taking care not to introduce any air bubbles. See
below for the appropriate solution level:
No stacking gel
(Continuous system.) Fill solution to just below the
top of the upper plate edge. If bubbles are trapped,
remove with a pipet or syringe. Introduce a comb (at
a slight angle) into each sandwich, taking care not to
trap air bubbles under the teeth.
2-gel sandwich
Pipet the solution into both sandwiches, filling each
to the same level below the notched edge.
Stacking gel
Fill solution to 3–4 cm below the top of the glass
plate. This height allows 1 cm of stacking gel below
the wells. Pour the gel and apply an overlay (see step
3). After the gel is set, prepare the stacking gel as
described in the next section.
2-D electrophoresis
(Discontinuous system) For the second dimension
resolving gel, fill solution to ~1.0 cm below the top of
the glass plate (leave extra space for a stacking gel, if
required). One centimeter allows enough space for the
first dimension IPG strip or tube gel and an agarose
seal. (While transferring, take care to avoid trapping
air between the tube gel and slab gel; seal the tube
gel into place with agarose in electrophoresis buffer.)
Note: Appendix A lists recipes
for the Laemmli gel system.

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3
If combs are in place, skip to step 4.
If no combs are in place, overlay the resolving gel
with a thin layer of water-saturated n-butanol, water,
or diluted gel buffer to prevent exposure of the top
surface of the gel solution to atmospheric oxygen.
Slowly deliver the overlay solution from a glass
syringe fitted with a 22-gauge needle. Apply the
solution near the spacer and allow it to flow across
the surface unaided.
4
Allow the gel to polymerize for a minimum of
one hour.
2.2.2. stacking gel
Pour the stacking gel before removing the sandwich
from the gel caster. Stacking gel resolution is optimal
when prepared just before electrophoresis.
1
Remove the overlay by rinsing the top of the gel
several times with distilled water. Invert the caster
to drain. To ensure a seamless contact between the
resolving and stacking gels, remove residual liquid by
blotting one corner with a lint-free tissue.
2
Calculate the stacking gel monomer solution volume.
3
Prepare the stacking gel monomer solution, deaerate
it, and add catalyst and initiator. Pour the stacking
gel onto the resolving gel with a disposable or Pasteur
pipet to a level about 2 mm from the top of the
glass plate.
4
Introduce a comb (at a slight angle) into the sand-
wich, taking care not to trap air under the teeth. Allow
a minimum of one hour for the gel to polymerize.

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2.2.3. gradient gels
Linear gradient gels can be poured in the gel caster.
For easy gradient mixing, we recommend using one of
the Hoefer SG series gradient makers. Gradient gels
are poured from the top of the caster with a cannula
if using the provided gel caster or from the bottom if
using a Hoefer multiple gel caster (see instructions
accompanying the caster). Once the gradient gel
polymerizes, a stacking gel is poured.
1
Assemble the glass plate assembly into the caster as
described in section 2.1.2.
2
Set up the monomer solution flow path. Run a length
of clear vinyl tubing through a peristaltic pump.
Attach one end of the tubing to the gradient maker
outlet port and the other end to a 20 cm cannula.
(The outside diameter of the cannula must be less
than the spacer thickness.) Place the cannula so
that it rests at the bottom of the sandwich, midway
between the spacers.
3
Prepare the monomer solution. Calculate the total
volume needed. Prepare one half of this volume of
higher and the other half of lower % acrylamide solu-
tion. (Optional: Add 15% sucrose or 25% glycerol
[final concentration] to the higher % solution to
improve layering.)
4
Pour the “light” solution into the reservoir chamber
(the chamber furthest from the inlet). Open the
stopcock long enough to displace air between the
chambers and then close. Pour the “heavy” solution
into the mixing chamber and place a stirring bar into
this chamber. Place the gradient maker on a magnetic
stirrer and begin stirring at a rate that does not
introduce bubbles in the solution.
Note: With Coomassie™ Blue,
it is possible to detect 1 µg in
a single band; with the more
sensitive silver stains, it is
possible to detect as little as
10 ng.
Fig 6. Pouring a gradient gel

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5
Mix the gradient. While the solution is stirring, begin
pumping (5–10 ml/min) from the mixing chamber and
immediately open the stopcock to the reservoir cham-
ber. Raise the cannula as liquid enters the sandwich,
keeping the tip at the gel surface.
6
Overlay each gel with a thin layer of water-saturated
n-butanol, water, or diluted gel buffer to prevent gel
exposure to oxygen. Slowly deliver the overlay solution
from a glass syringe fitted with a 22-gauge needle.
Apply the solution near the spacer and allow it to flow
across the surface unaided.
7
Allow the gel(s) to polymerize for a minimum of one
hour. After polymerization, pour off the overlay and
rinse the gel surface several times with distilled water.
8
Prepare the stacking gel monomer solution, pour the
stacking gel and introduce a comb (at a slight angle)
into the sandwich, taking care not to trap air under
the teeth. Allow a minimum of one hour for the gel
to polymerize.

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2.3. Sample preparation
The amount of sample loaded depends on the
thickness of the gel, the sensitivity of the detec-
tion method used, and the amount of sample
expected in each band. In a continuous buffer
system, the protein sample should be relatively
concentrated because no stacking gel is used.
In a discontinuous buffer system, the zone into
which each molecular species migrates is sharp-
ened by the stacking gel, so the sample need not
be as concentrated.
1
Prepare the wells. Remove the comb by gently
rocking it side to side and then lifting it straight
up to avoid damaging the well walls. Carefully rinse
each well with electrophoresis buffer to remove
unpolymerized acrylamide and then drain by inverting
the gel sandwich (or caster). Fill each well with
electrophoresis buffer.
2
Prepare the sample. Increase liquid sample density
with 10% glycerol or sucrose. Add a tracking dye such
as phenol red, bromophenol blue, or pyronin Y.
For SDS protein gels, use 2X treatment buffer to
denature both liquid and dry samples in a test tube:
To liquid protein samples, add an equal volume of
2X treatment buffer.
To dry protein samples, add equal volumes of 2X
treatment buffer and deionized water to achieve the
desired concentration.
Heat the tube in boiling water for 90 seconds, then
allow to cool to room temperature. Treated samples
can be stored at -40 to -80 °C for future runs.
Heat membrane proteins to 60 °C for 20 minutes.
Store unused sample at 4 °C.
Table 2. Well volume (µl) per
1 mm depth for each comb size
Comb thickness
(mm)
No. of wells 0.75 1.0 1.5
10 6.2 8.3 12.4
12 5.8 — 11.5
15 4.3 5.7 8.6
20 3.1 4.1 6.2
28 2.1 2.7 4.1

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Note: Before the first use,
disassemble the unit and
wash with a dilute solution
of a laboratory detergent and
rinse thoroughly, first with
water and then distilled water.
Note: To help hold the gasket
against the upper buffer
chamber, dab a small amount
of GelSeal at each end of the
gasket only and then install.
Important! A smooth fit
between the sandwich and
gasket is essential for a
good seal.
2.4. Final assembly
1
Rinse both buffer chambers with water and distilled
water thoroughly before each use.
2
Install the gel sandwich in the lower buffer chamber
Release the sandwich from the caster by removing
both cams. Clean away any gel adhering to the
exterior of the gel sandwich. Install the sandwich
in the lower buffer chamber, clamp screws facing
toward the leads.
3
Carefully fill each sample well with electrophoresis
buffer, then underlay prepared sample into the
wells using a fine-tipped microsyringe or gel loading
pipet tip.
4
Attach the upper buffer chamber to the gel sandwich
Invert the upper chamber and press the slotted gasket
into the grooves for a precise fit.
Proceed with care so that the samples are not
disturbed: Lower the upper chamber onto the gel
sandwich. Install the cams, ridge pointing down, into
the cam holes as shown on page 17. Simultaneously
turn one cam clockwise and the other counterclock-
wise a full 180° to secure the assembly.