Hoefer TE 70 User manual

user manual
TE70 and TE77
um TE70&77-IM/Rev. H0/06-04
Hoefer TE 70 and TE 77
semi-dry transfer units

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Page finder
Semi-dry Transfer Unit
function and description ...............................1
Unpacking ...............................................2
Specifications ..........................................2
Important information ...............................3
Operating instructions ..................................4
Care and maintenance ................................10
Troubleshooting ..........................................11
Electrotransfer notes ...................................13
Bibliography ..............................................15
Ordering information ..................................16

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English
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Safety warnings and precautions
Important user information
Please read this entire manual to fully understand the safe
and effective 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.
Hoefer, Inc. reserves the right to make changes in the specifi-
cations without prior notice.
Warranty and liability
Hoefer, Inc. guarantees that the product delivered has been
thoroughly tested to ensure that it meets its published speci-
fications. 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 Hoefer, Inc.
Hoefer, Inc. shall in no event be liable for incidental or conse-
quential 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.
Renseignements importants
d’utilization
Pour une bonne compréhension et une utilisation en sécurité
maximale, il convient de lire entièrement ce manuel.
Dans la documentation qui accompagne l’instrument un point
d’exclamation dans un triangle équilatéral a pour but d’attirer
l’attention de l’utilisateur sur des instructions importantes de
fonctionnement ou de maintenance.
Le symbole de l’éclair dans un triangle équilatéral a pour
objet d’attirer l’attention de l’utilisateur sur un danger
d’exposition à la haute tension.
Garantie et responsabilité
Hoefer, Inc. garantit à l’utilisateur que le produit livré a subi
avec succès tous les essais prévus pour s’assurer qu’il est
conforme aux spécifications et normes en vigueur. La garantie
incluse dans les conditions de livraison n’est valable que si le
produit a été installé et utilisé conformément aux instructions
fournies par Hoefer, Inc.
La société Hoefer, Inc. ne sera en aucun cas responsable de
tout dommage causé directement ou indirectement par toute
utilisation incorrecte ou non approuvée du produit ou décou-
lant de cette utilisation, y compris toute perte de bénéfice ou

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de recettes, toute perte de perspectives commerciales, tout
empêchement d’utilisation et tout autre risques ayant
un rapport avec l’utilisation du produit, mais sans aucune
limitation quant à la nature de ces dommages.
Wichtige benutzerinformationen
Für ein vollständiges Verständnis und eine sichere Hand-
habung dieses Produktes ist es notwendig, daß der Benutzer
dieses Handbuch vollständig durchliest.
Ein Ausrufezeichen in einem gleichseitigen Dreieck soll
den Benutzer auf die Anwesenheit wichtiger Betriebs- und
Wartungsanweisungen in der dem Gerät beiliegenden Doku-
mentation hinweisen.
Ein Blitzsymbol in einem gleichseitigen Dreieck soll den
Benutzer auf die Gefahr anliegender Hochspannungen
hinweisen.
Hoefer, Inc. behält sich das Recht vor, die Spezifikationen
ohne vorhergehende Ankündigung zu ändern.
Gewährleistung and haftung
Hoefer, Inc. garantiert, daß das gelieferte Produkt sorgfältig
auf die Einhaltung der veröffentlichten Spezifikationen
getestet wurde. Die in den Lieferbedingungen näher erläuter-
ten Gewährleistungsansprüche gelten nur dann, wenn das
Produkt gemäß den von Hoefer, Inc. gelieferten Anweisungen
installiert und benutzt wurde.
Hoefer, Inc. übernimmt keinerlei Haftung für Schäden oder
Folgeschäden, einschließlich, aber nicht begrenzt auf Gewin-
neinbußen, Einkommensverluste, entgangene Geschäftsab-
schlüsse, Verlust der Gebrauchsfähigkeit oder andere Verluste,
die wie auch immer durch eine fehlerhafte oder unsach-
gemäße Verwendung des Produkts verursacht wurden.
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Información importante para el usuario
Para comprender el producto y utilizarlo con seguridad es
necesario leer este manual en su totalidad.
El signo de admiración en un triángulo equilátero en el
manual, advierte al usuario sobre la presencia de instruccio-
nes importantes de operación y mantenimiento del aparato.
El símbolo del rayo en un triángulo equilátero alerta al
usuario sobre el riesgo de exposición a altas tensiones.
Hoefer, Inc. se reserva el derecho a modificar las especifica-
ciones sin previo aviso.
Garantía y responsabilidad
Hoefer, Inc. garantiza que el producto entregado ha sido
probado a fondo para comprobar el cumplimiento de las espe-
cificaciones publicadas. La garantía incluida en las condicio-
nes de entrega sólo es válida si el producto se ha instalado
y utilizado de acuerdo con las instrucciones entregadas por
Hoefer, Inc.
Hoefer, Inc. no será responsable, bajo ningún concepto,
de daños directos o indirectos, incluyendo sin limitación la
pérdida de beneficios, la pérdida de ingresos, la pérdida de
oportunidades de negocio, la pérdida de utilización y otras
consecuencias relacionadas, cualquiera que sea la causa, que
se deban a la utilización defectuosa e incorrecta del producto.
Informazioni importanti per l’operatore
Per un utilizzo sicuro del prodotto, leggere attentamente
l’intero contenuto del presente manuale.
Il punto esclamativo all’interno di un triangolo equilatero
indica all’operatore la presenza di importanti istruzioni di
funzionamento e manutenzione nella documentazione allegata
al prodotto.
Il simbolo del fulmine all’interno di un triangolo equilatero
indica all’utente la presenza di un rischio di esposizione ad
alte tensioni.
Hoefer, Inc. si riserva il diritto di apportare modifiche ai dati
tecnici senza preavviso.
Garanzia e responsabilitá
Hoefer, Inc. garantisce che prima della consegna il prodotto è
stato collaudato a fondo per soddisfare i requisiti specificati.
La garanzia inclusa nelle condizioni di consegna risulta valida
solamente se il prodotto è stato installato ed utilizzato nel
rispetto delle istruzioni fornite da Hoefer, Inc.
Hoefer, Inc. non potrà essere ritenuta responsabile di inci-
denti o danni consequenziali, inclusi’ma non limitati’a perdite
di profitti, mancato guadagno, perdite di affari, difetti di
funzionamento e relative esposizioni, dovuti ad un utilizzo
non corretto del prodotto.
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Semi-dry Transfer Unit
function and description
The TE 70 and TE 77 semi-dry transfer units
rapidly transfer proteins from polyacrylamide gels
onto a membrane by means of a low current and
low voltage electrotransfer with minimal Joule heat-
ing. Most transfers are complete in one hour or less.
The smaller TE 70 unit transfer surface is 14 × 16 cm,
and the larger TE 77 transfer surface is 21 × 26 cm.
Several small gels of the same thickness can be laid
side-by-side or two larger gels can be stacked into
a carefully constructed multi-layered transfer stack.
A plastic mask may be used to prevent current from
bypassing the gel stack.
The base and cover body are made of inert Noryl®
plastic. The base acts as a reservoir for buffer that
may leak during the transfer. The anode in the base
is platinum coated and the cathode in the cover is
stainless steel. A safety interlock switch breaks the
circuit when the cover is removed.
Cover
(contains the cathode)
Safety-interlock lead
Safety-interlock housing
Base
(contains the anode)
Color-coded leads
Mask
Guides, (3)
Fig 1. Semi-dry transfer unit
main components.
A power supply with the follow-
ing rating is required but not
included:
TE 70: up to 200 mA, 50 V
TE 77: up to 450 mA, 50 V

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Unpacking
Unwrap all packages carefully and compare
contents with the packing list, making sure all
items arrived. If any part is missing, contact
Hoefer, Inc. 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.
Specifications
TE 70
Max. wattage 10 W
Max. voltage 50 V~
Max. amperage 200 mA
Gel size up to 14 × 16 cm
TE 77
Max. wattage 22.5 W
Max. voltage 50 V~
Max. amperage 450 mA
Gel size up to 21 × 26 cm
Environmental
operating conditions Indoor use: 4–40 °C
Humidity up to 80%
Altitude up to: 2000 m
Installation category: II
Pollution degree: 2
Dimensions (w × d × h) 32 × 25 × 9 cm
(12.6 × 9.9 × 3.6 in.)
Product certifications EN61010–1, UL3101–1,
CSA C22.2 1010.1, CE
This declaration of confor-
mity 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.
• connected to other CE
labeled instruments or
products recommended or
approved by Hoefer, Inc.

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Important information
• The electric components in the safety interlock
housing of the transfer unit base must not become
wet. Do not immerse any part of the unit in water.
Rinse only the electrodes with distilled water before
use. (Refer to the Care and maintenance section for
cleaning instructions.)
• Be sure to apply enough buffer-soaked sheets of
blotting paper under the membrane and over the
gel to prevent the stack from drying out. Irreparable
damage to the unit will result if the stack dries out
and the unit is allowed to overheat.
• 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.
Informations importantes
• Les piéces électriques contenues dans la chambre
de sécurité, à la base du unité de transfert semi-sec,
ne doivent pas être mouillées. Ne submerger aucune
piéce de l’instrument. Rincer seulement les élec-
trodes avec de l’eau distillée avant l’utilisation. (Voir
la section Care and maintenance pour l’entretien).
• Appliquer du buvard suffisamment saturé de
tampon sous la membrane et sur le gel afin d’éviter
à la pile de buvards de se dessécher. Des dommages
irréparables à l’appareil peuvent résulter si la pile
de buvards est séche et l’instrument surchauffe.
• Si l’instrument n’est pas utilisé en conformité avec
les recommandations du fabriquant, les dispositifs
de sécurité de cet appareil pourraient être inéffi-
caces si l’instrument n’est pas utilisée.
• Seulement les accessoires et piéces détachées
approuvés ou fornis par Hoefer, Inc. sont recom-
mandés pour l’utilisation, l’entretien et réparation
de cet appareil.
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Note: See Electrotransfer
notes on page 11 for transfer
tips as well as two buffer reci-
pes and references.
Operating instructions
To transfer proteins, prepare the unit and trans-
fer stack components, assemble the stack, and
connect the leads to a power supply. Then run the
transfer for the required amount of time. Each
step is described below.
Assemble the transfer stack

Prepare the unit by rinsing the anode and cathode with
distilled water, taking great care to keep water away
from the safety interlock housing and leads.

Prepare the gel
Cut away the wells and/or stacking gel section. Equili-
brate the gel in transfer buffer if required. Measure
the gel.

Cut a mask to the proper size
Cut an opening in the solid mask to a size slightly
smaller than the gel.
The required opening is centered and 2 mm smaller
than the gel on all sides. Because the surface is slick
and hard, take care when cutting mask with a tool
such as a mat knife.
Place the mask in the base of the unit, centering the
opening.

Prepare the blotting paper
For each gel, cut at least 6 pieces of blotting paper
the same size as the gel or slightly smaller.
Note: Always wear gloves
when handling membranes to
avoid leaving fingerprints.

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Gauge the thickness or number of blotting paper
layers according to the amount of buffer required; up
to 250 ml of buffer may be required for larger gels
or for transfers of 60 minutes in order to prevent the
stack from drying out or the buffer from becoming
depleted. Saturate at least 3 pieces of the blotting
paper with transfer buffer. One by one, center each
sheet over the cutout on the mask and remove all
trapped air by rolling a clean pipet from the center
toward the edges. Check that the blotting paper
completely covers the mask cutout and slightly over-
laps the mask edges on all sides.

Prepare the membrane
For each gel, cut 1 membrane the same size as
the gel or slightly smaller. (A larger membrane may
contact an electrode panel, creating a pathway by
which current can bypass the gel.)
Pre-wet nitrocellulose or nylon membranes with
distilled water. Pre-wet PVDF or other hydrophobic
membranes with methanol. Then soak all membrane
types in transfer buffer for 2–5 minutes.

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
Complete the stack
a) Place the pre-wet membrane onto the stack of
blotting paper.
b) Place the gel on the membrane. Note: Proteins
bind to the membrane as soon as contact occurs,
so it is important to place the gel correctly on the
first try.
c) Cover the gel with several layers of saturated
blotting paper.
Multiple gels: Either lay gels of the same thickness
side-by-side (the position of the gels must coincide
with cutouts in the mask), or stack 2 sandwiches
layered as shown on the next page.
Fig 2. Transfer stack for a single gel.
Mask (opening
approx. 2 mm
smaller than
the gel on all
sides)
Blotting paper
(2–3 sheets)
Blotting paper
(2–3 sheets)
Gel
Membrane
These stack compo-
nents are the same
size as the gel or
slightly smaller
Note: Take care to place the gel correctly on the first try
because proteins begin to transfer immediately; once
transfer has begun, moving the gel will distort results or
cause “shadow” bands on the blot.
Important! Stack each layer
with care, with edges parallel.
As each layer is added, remove
all air pockets by rolling a
clean pipet from the center to
the edges. Add a few drops of
buffer to trouble areas to help
remove air pockets.

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Fig 3. Transfer stacks for gels
placed side-by-side.
Check electrical continuity within
each stack: several sheets of
buffer-soaked blotting paper
must cover each gel, and each
stack must fully contact both
electrodes.
Note: There must be no electrical
contact between the two stacks.
Mask
Blotting
paper, (2–3
sheets)
Blotting
paper, (2–3
sheets)
Gel
Membrane
If two gels are stacked, separate them with porous
cellophane — not plastic wrap! (Cellophane permits
electric current to pass but stops proteins.) Cut the
cellophane slightly smaller than the gel and wet with
transfer buffer. Several sheets of buffer-soaked blot-
ting paper on each gel provides electrical continuity.
Note: When transferring multi-
ple gels, transfer efficiency
depends on such factors as
gel thickness, gel position
in the stack, transfer buffer,
membrane type, and, most
importantly, the characteris-
tics of the protein. The gel
closest to the anode generally
transfers the most completely,
so it is preferable to lay gels
side-by-side rather than stack-
ing them.
Blotting paper,
(2–3 sheets)
Blotting paper,
(1 sheet)
Cellophane
Mask
Gel
Membrane
Blotting paper,
(2–3 sheets)
Gel
Membrane
Fig 4. Transfer stack for stacked
gels.
Separate the stack for each gel
with a sheet of cellophane.

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
Slide the 3 guides on the cover into the 3 grooves in
the transfer unit base. Do not remove the cover until
after the transfer is complete in order to prevent stack
components from sliding out of alignment.

If transferring more than one gel, place a 1 kg weight
on the cover to improve the electrical continuity of the
stack. Note: A weight of more than 1 kg may press
too much buffer out of the blotting paper, which will
adversely affect the transfer efficiency.
Transfer

Set the power supply high voltage limit to 50 V
If possible, set the voltage limit before connecting the
transfer unit. Use only an approved power supply such
as the Hoefer PS2A200.

The power supply should be switched off and both the
current and voltage controls turned to zero
Plug the short safety-interlock lead from the cover
into the jack on the base. Then plug the color-coded
leads from the base of the transfer unit into the power
supply jacks, matching red to red and black to black.
Do not reverse polarity. (Red is positive [+] and black
is negative [–].)

Set the power supply current
The maximum current setting must not exceed
0.8 mA/cm2 of the gel surface. If transferring several
layers of gels, a longer transfer time may be required.
Use the graph to quickly find the maximum current
setting for your gel size, or calculate the gel area
(cm2) and multiply it by 0.8 mA/cm2.
Note: Staining the gel(s) for
residual protein gives an indi-
cation of the completeness of
transfer.
Note: Rewet dried membranes
in the appropriate wetting
buffer prior to processing.

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cm2
Gel
Size
maximum mA
14 × 24
14 × 16
8 × 10
8 × 7
Fig 5. Recommended maximum
current settings for different gel
sizes.

If available, set the power supply timer
Most transfers are complete within one hour, but
larger proteins, proteins from native gels, and thicker
gels may require an additional 1 hour of transfer time.
The optimum transfer time for each protein and gel
system must be determined empirically.
When developing a new protocol, it is important to
monitor the voltage closely. It should not exceed 50 V.
High initial voltage (20 V or more) indicates inap-
propriate ionic strength; check the buffer concentra-
tion and quality. If the voltage increases more than
10 V during the run, the stack may be drying out and
scorching may result, which can damage the sample
or the transfer unit. Another indicator of buffer deple-
tion is increasing temperature; feel the top of the
cover for excessive heat periodically, especially after
one hour. If drying is indicated by either high voltage
or increased temperature, stop the transfer and add
more layers of buffer-saturated blotting paper.
Note: Generally, smaller frag-
ments transfer more quickly
than larger ones.
Note: After 1 hour of transfer
time, switch off the power
supply and re-wet the stack.
Transfer can then proceed for
an additional hour.

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After the transfer is complete

Turn off the power supply and disconnect the leads
from the power supply.

Unplug the safety-interlock lead between the cover
and the base of the unit. Remove the cover slowly
because the stack may adhere to it.

Remove and dispose of the blotter paper.

Remove the gel(s). Optional: Stain for residual protein.

If desired, label the gel contact side of the membrane
with a soft pencil.

Remove the membrane(s) from the stack with blunt
forceps. Process the membrane following instructions
accompanying your protocol or allow the membrane to
air dry prior to storage.

Remove the remaining blotter papers and dispose.
Remove the mask. Rinse the unit according to the
Care and maintenance instructions.
Care and maintenance
Rinse the cathode in the cover and the anode in
the base with distilled water. Let the unit air dry
completely. If using radioactive reagents, decon-
taminate the unit with a cleaning agent such as
Decon™ 90 or Contrad™ 70. Never use abra-
sive cleansers.
• Do not autoclave or wash
the unit in a dishwasher.
• Do not wet the safety-inter-
lock housing or leads.
• Do not immerse the unit in
water!

• p11
problem solution
Incomplete transfer
Blank or faint
areas on the
membrane
Remove trapped air pockets between the gel and membrane during
stack assembly.
Reduce the current setting to minimize the amount of gas produced
by the electrolysis of buffer.
Use buffer with a lower ionic strength.
Molecules do
not migrate out
of gel
Check all electrical connections. Check that current is flowing by
plugging a multi-meter into the circuit and comparing the meter
reading with the power supply reading.
Check that the buffer pH is close to the intended pH. Most buffers
should not be titrated; make fresh buffer.
Use 3.5 mM SDS (0.1%) in the transfer buffer.
Add several more sheets of buffer-saturated blotting paper to each
side of the gel sandwich so that more buffer is present during the
transfer.
Do not use staining or fixing agents on the gel before transfer.
Use a thinner gel.
Reduce the gel acrylamide concentration.
Increase the transfer period. Large fragments may require an addi-
tional hour.
If using a non-nitrocellulose membrane, avoid including methanol in
the transfer buffer or reduce the amount to the minimum possible.
Use reagent-grade chemicals.
Increase the net charge on the protein by using a transfer buffer
with a different pH. Lower pH (<6–7) increases the positive charge
on proteins; higher pH (>6–7) increases the negative charge on
proteins.
Troubleshooting

• p12
Smeared or diffuse
band patterns
If equilibrating before the transfer, shorten or eliminate the equilibra-
tion time and/or equilibrate under coldroom conditions.
If the transfer buffer contains ≥10% methanol, equilibrate the gel in
transfer buffer for 30 minutes to allow it to shrink before assembling
the stack. Note: Large proteins may not migrate as readily once the
pore size is slightly reduced.
Take care that the gel does not shift once it contacts the membrane.
If excess heating occurs during the transfer, note if the stack is
drying out. On subsequent transfers, either shorten the transfer
time, reduce the current, or increase the number of buffer-soaked
blotting papers in the stack.
Check that the preferred binding surface of the membrane (if any)
contacts the gel.
Uneven band
transfer
The blotting paper and membrane must be the same size as the gel
or 1–2 mm smaller. Larger sizes will provide an electrical path for
current to bypass the gel.
Inefficient binding
to membrane
Chemical
parameters
Fix or crosslink the molecule onto the membrane according to the
requirements of the nucleic acid, protein, or membrane type.
Prepare transfer buffer without SDS. (SDS can improve transfer effi-
ciency but can interfere with protein binding to a PVDF membrane.)
Add 10–20% methanol to the transfer buffer to enhance binding to
nitrocellulose.
Membrane
parameters
Use a membrane with a smaller pore size (0.10–0.20 µm) if
proteins pass through the membrane.
Place a membrane both over and under the gel to capture any
proteins migrating in the opposite direction.
Check if too much sample is available for the binding surface area
by placing two membranes instead of one. If “blow through” occurs,
reduce the sample load.
Wear gloves when handling membranes.
Store membranes at ambient temperature and out of direct sunlight.

• p13
Electrotransfer notes
• Run the transfer as soon as possible after electro-
phoresis to minimize protein diffusion within the gel.
• Stacked gels must all be the same size.
• Limit transfers to two hours or less, rewetting the
stack after 1 hour.
• The recommended methanol concentration for
different membrane types are:
membrane type methanol %
Charged nylon 0
Nitrocellulose 20
PVDF 15
• Use a buffer with low ionic strength such as one of
the two listed below to prevent overheating. Use the
CAPS buffer when Tris cannot be used (e.g. peptide
sequencing). CAPS can improve transfer because of
its effect on the charge of the protein (see Matsud-
aira, 1987).
Towbin buffer
(25 mM Tris, 192 mM glycine, 20% v/v methanol,
pH 8.3, 1 liter)
Tris (FW 121.1) 25 mM 3.0 g
Glycine (FW 75.07) 192 mM 14.4 g
SDS* (FW 288.4) 0.1% (3.5 mM) 1.0 g
Dissolve in 600 ml distilled water. Add methanol as
required†. Bring to 1 liter with distilled water.
Do not adjust the pH, which should be between 8.2
and 8.4. Optional: Chill before use.
*Optional: Adding SDS can improve transfer efficiency.
† Depending on the membrane type selected (see table
above), adding methanol can improve transfer results.
Note: Buffers containing methanol may deteriorate if
stored for long periods —add methanol just prior to trans-
fer.

• p14
CAPS buffer, 1X
(10 mM CAPS, pH 11.0, 1 liter)
CAPS (FW 221.3) 10 mM 2.2 g
[3-(cyclohexylamino)-1-propanesulfonic acid]
Dissolve in 600 ml distilled water, adjust to pH
11.0 with conc. NaOH. Adjust volume to 1.0 liter.
• For a 3-buffer system, refer to Kyhse-Anderson, J.
(1984).
• Transfer efficiency varies depending on the gel
concentration, which can be optimized. For more
information, refer to Smejkal and Gallagher (1994).