Thermo Scientific OWL HEP-1 Operating instructions

Semidry Electroblotter
Models HEP-1 and HEP-3
Operating and Maintenance Manual 7007332 Rev. 0
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Thermo Scientific
MANUAL NUMBER 7007332
0 -- 5/1/12 Transfer to Marietta (was The Panther 3/2003) ccs
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Preface
Semidry Electroblotter i

Thermo Scientificii Semidry Electroblotter
Preface
Important Read this instruction manual. Failure to read, understand and follow the instructions in this manual
may result in damage to the unit, injury to operating personnel, and poor equipment performance. s
Caution All internal adjustments and maintenance must be performed by qualified service personnel. s
Warning To avoid the risk of personal shock, always disconnect the gel box from the power supply. Further,
the power supply must be equipped with a shut-down-on-disconnect circuit. Running conditions for this unit
should not exceed the name plate readings found on the lower buffer chamber. s
This system is designed to meet IEC 1010-1 safety standards (IEC 1010-1 is an internationally accepted
electrical standard for laboratory instruments).
Statement of Proper Use: Use this product only for its intended purpose as described in this manual. Do not
use this product if the power leads are damaged or if any of its surfaces are cracked.
Material in this manual is for information purposes only. The contents and the product it describes are subject
to change without notice. Thermo Fisher Scientific makes no representations or warranties with respect to this
manual. In no event shall Thermo Fisher Scientific be held liable for any damages, direct or incidental, arising
out of or related to the use of this manual.
©2012 Thermo Fisher Scientific. All rights reserved.

Thermo Scientific Semidry Electroblotter iii
Preface
Important operating and/or maintenance instructions. Read the accompanying text carefully.
Potential electrical hazards. Only qualified persons should perform procedures associated with this
symbol.
Equipment being maintained or serviced must be turned off and locked off to prevent possible injury.
Hot surface(s) present which may cause burns to unprotected skin, or to materials which may be
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Marking of electrical and electronic equipment, which applies to electrical and electronic equipment
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after 13 August 2005.
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European Country, and this product should be disposed of or recycled through them. Further
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4Always use the proper protective equipment (clothing, gloves, goggles, etc.)
4Always dissipate extreme cold or heat and wear protective clothing.
4Always follow good hygiene practices.
4Each individual is responsible for his or her own safety.

Thermo Scientificiv Semidry Electroblotter
Preface
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Semidry Electroblotter vThermo Scientific
Table of Contents
General Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1-1
Setting Up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-1
Materials Needed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-1
Setting Up the Blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-2
DNA Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-3
Gel Sandwich Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2-4
Using the System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-1
Running the Blot . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-1
Transfer settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-1
Running Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3-2
Factors That Affect Transfer Efficiency . . . . . . . . . . . . . . . . . . . . . .3-2
Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4-1
Technical Tips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-1
Semidry Blotting - General References . . . . . . . . . . . . . . . . . . . . . .5-2
Recipes for Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5-3
Care and Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6-1
Optional Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7-1
Section 1
Section 2
Section 3
Section 4
Section 5
Section 6
Section 7

Semidry Electroblotter 1-1Thermo Scientific
Section 1 General Information
The Thermo Scientific semi-dry electroblotter offers rapid transfer of
proteins or nucleic acid molecules from polyacrylamide or agarose gels to
membranes. Solid plate platinum/titanium and stainless steel electrodes are
highly conductive and allow transfer at low voltages without external
cooling systems. Plate electrodes also provide a uniform electric field for
efficient, even transfers.
Before starting, unpack the unit and inventory your order. If any parts are
missing, contact Technical Services immediately. You have 30 days from
date of shipment to make any shipment claims.
Item Description . . . . . . . . . . . . . . . . . .Catalog No.
Complete System . . . . . . . . . . . . . . . . . . . . . .HEP-1
Transfer Area . . . . . . . . . . . . . . . .20cm W x 20cm L
Footprint . . . . . . . . . . .25cm W x 25cm D x 5cm H
Complete System . . . . . . . . . . . . . . . . . . . . . .HEP-3
Transfer Area . . . . . . . . . . . . . . . .35cm W x 45cm L
Footprint . . . . . . . . . . .40cm W x 51cm D x 5cm H

1-2 Semidry Electroblotter Thermo Scientific
Section 1
General Information
Heavy Duty Knobs
HEP-1 (3)
HEP-3 (4)
Power Supply Leads
Lid
Anode
Cathode
Base
Figure 1-1. Exploded View
Table 1-1. Parts List
• 1 base with stainless steel cathode
• 1 lid with platinum/titanium anode
• 1 attached pair of power supply leads
• 3 knobs for the HEP-1, 4 knobs for the HEP-3

Semidry Electroblotter 2-1Thermo Scientific
Section 2 Setting Up
Once the proteins (or nucleic acids) in a sample aliquot have been
separated on a slab gel, the resulting bands may be transferred to a solid
support membrane. The primary reason for this type of blot is one of
localization and secondarily, concentration of discrete bands. Although
many have used alternative cross linking agents such as DATD
(N,N'dihydroxyethylene-bis Acrylamide) to allow for the accessibility of
gel bound proteins, this still represents an impediment to radio
enumeration due to the quenching by the gel matrix itself.
The most common solid support membrane is nitrocellulose. A second
type of membrane is PVDP (Polyvinylidene difloride) which is generally
used when a transferred protein is to be sequenced, additionally, it has a 2x
binding capacity. Also used for nucleic acid capture are Nylon membranes.
In either case, the proteins are transferred from the gel to the matrix in an
electric field perpendicular to the gel (initial running direction).
Tris based buffers are employed in the transfer. Methanol and SDS are
modifiers often use in protein transfer buffer. These components however
are antithetical in their effects both in terms of movement and adsorption.
Methanol restricts protein movement from the gel but is often required to
support the ionic nature of protein to nitrocellulose binding. SDS aids in
protein elution, but can also inhibit binding of small molecular weight
proteins (Mozdzanowski, J., High yield electroblotting, Electrophoresis,
1992, Vol l3.,p.59-64).
In order to use this blotting device, you will need:
Power supply - Blotting requires a power supply that can operate at a fairly
high current setting and low voltage. If an inappropriate power supply is
used, the power supply may blow a fuse, shut itself off, display a no load or
short load message or even have a short circuit. It is very important to be
sure that the power supply you will be using will work with this device.
Some power supplies that will work with this device are Catalog No. OSP-
135, OSP-300, EC Apparatus EC135 and EC570, and Bio Rad's
PowerPac 200. If your power supply is not among those listed, contact the
manufacturer of your power supply to determine if it will work for tank
blotting applications. Contact Technical Services for power supply
recommendations if you do not have an appropriate power supply.
Materials Needed

2-2 Semidry Electroblotter Thermo Scientific
Section 2
Setting Up
Materials Needed
(continued)
Blotting buffer - The most commonly used buffer for protein blotting
from polyacrylamide gels is Towbin buffer. Small amounts of buffer may be
needed for equilibrating the gel and membrane prior to blotting, in
addition to the buffer in the transfer tank. Should be cooled to 40°C.
Filter paper - Sometimes called blotting paper, it used in the blotting
sandwich.
Blotting membrane - Nitrocellulose and PVDF (Polyvinylidene difloride)
can be used for proteins, while charged Nylon membranes can be used for
nucleic acids. The choice depends upon the user's preference and
sometimes the detection method to be used.
There are three types of gels, each requiring a different handling procedure
before they can be added to the blotting sandwich. Protein and agarose
gels set up are arranged together, with sequencing gel transfer protocol
following separately.
DNA/RNA: If these gels were not run in IXTBE, they should be
equilibrated for 10 minutes in this buffer.
Protein Gels:
After electrophoresis, remove the gel assembly from the apparatus and
remove the spacers.
Open the gel cassette by gently rocking a spatula between the plate, forcing
separation of the plate from the gel. The gel will normally remain affixed to
the bottom plate. Remove the top (notched) plate by slowly lifting it from
the side with the inserted spatula and gradually increasing the angle until
the plate is completely separated from the gel.
If the gel sticks to the top plate in an isolated spot, a stream of water from
a squirt bottle can be sprayed at the spot to aid separation.
Remove the gel from the remaining plate. Tip the plate up side down, and
start one edge, and allow it to roll off into transfer buffer. Alternatively,
place the plate with the gel attached, into transfer buffer.
Incubate the gel in transfer buffer for 15 min. with gentle agitation. If the
gel is on the plate, it will become loose during this step.
Setting Up the Blot

Semidry Electroblotter 2-3Thermo Scientific
Section 2
Setting Up
DNA Gels Preparing a sequencing gel for transfer on the HEP-3
1. Remove the gel cassette from the gel box and place under cold,
running tap water until the surfaces of both glass plates are cool. This
facilitates handling of the gel and prevents the gel from "curling" as it
cools once it is removed from between the glass plates.
2. Place the glass plate sandwich, with the notched or shorter plate facing
up, flat on paper towels on the lab bench and remove any excess liquid
and remaining tape (or binder clips).
3. Remove one side spacer (wearing protective gloves) and insert a long
metal spatula between the glass plates where the spacer had been
present. Gently rock the spatula, forcing separation of the plate from
the gel. The gel will normally remain affixed to the bottom (not
notched) plate. Remove the top (notched) plate by slowly lifting it
from the side with the inserted spatula and gradually increasing the
angle lmtil the top plate is completely separated form the gel. If the gel
sticks to the top plate in an isolated spot, a stream of water from a
wash bottle can be sprayed at the spot to aid separation. Occasionally,
a gel sticks to the top plate instead of the bottom plate. In this case,
flip the gel sandwich over and follow the same procedure.
4. Once the plates are separated, remove the second side spacer along
with any extraneous bits of acrylamide around the gel. If excess water
was utilized to aid in the separation of the gel from the glass plate, use
a paper towel to absorb the excess liquid. Using a piece of dry blotting
paper(Whatman 3MM chromatography paper, 46 x 57cm), gently roll
it onto the gel beginning at one end and working slowly towards the
other end. Care should be taken to prevent air bubbles from forming
between the paper and the gel. Once the blotting paper is in position,
set another glass plate on top of the paper. Flip the entire sandwich
over. The gel should now be resting on top of the filter paper between
the two pieces of glass. Position the sandwich with one long side of the
glass hanging about one-third of the way off of the counter top. Slowly
push the bottom plate back toward the counter top, holding the top
plate in place, which will allow the blotting paper and gel to peel away
from the top piece of glass. The weight of the gel and blotting paper
causes the gel to slowly peel away from the top plate. Continue this
process until the gel is almost entirely peeled away from the glass. A
small portion of the gel may still be attached to the glass; in this case, a
small stream of water from a wash bottle can be used to aid in removal
of the gel.

5. An alternative procedure at this step is to peel the paper, with the gel
attached, from the glass plate upon which it rests. Beginning at one
end of the gel, slowly lift and peel back the paper with the gel attached.
As before, a stream of water from a wash bottle can be sprayed at any
spot to aid separation of the gel from the glass plate.
6. Place the gel resting on the Whatman, 3MM paper flat on a lab bench.
7. Wearing gloves, cut a piece of nylon membrane the size of the gel and
blotting paper. Briefly (but thoroughly) wet the membrane in 0.5 x
TBE in a shallow tray, being careful not to crease the membrane.
Holding the membrane by two corners, allow the excess liquid to drip
from the membrane into the tray.
1. Wearing gloves, cut the membrane to the size of the gel and blotting
paper.
2, Mark the membrane, to indicate the side to which the samples will be
on. This is important in the event that any successive probe is negative,
and to indicate sample orientation. This can be done by either clipping
a comer of the membrane or using a ball point pen. Clip the same
comer until you retire.
3. Wet the membrane according to its manufactures recommendations,
followed by a quick equilibration in transfer buffer. It is often helpful
to have all the filter paper and membranes sitting in transfer buffer as
you start to build the blotting sandwich.
4. With this device, the transfer is in the upward direction as shown in
the figure below, and the blotting sandwich is built upward, upon the
mirrored stainless steel plate of the bottom half of the blotter.
5. Lay three filter pads, one after the other down. Each should be soaking
wet with buffer. In fact, the lower chamber may filled with buffer as
you build the blotting sandwich.
6. Add a few mL of buffer to the filter pad, and gently layer the gel.
Beginning at one end of the filter paper, align the gel with the paper's
edge, and slowly lower the other end, driving out any bubble.
7. Wearing gloves, gently smooth out any bubbles by forcing them to the
closest edge of the gel. Test tubes and pipettes many also be used for
this purpose.
2-4 Semidry Electroblotter Thermo Scientific
Section 2
Setting Up
DNA Gels (continued)
Gel Sandwich
Assembly

8. Alternatively, you can place the filter paper in a box with buffer, toss in
the gel, and drain the box until the gel falls to the pad. Add a few mL
of buffer to the filter pads already in the bottom chamber. Then pick
up the paper and gel and layer them on the filter stack.
9. Add a few mL of buffer to the gel, and gently layer the membrane, as
you did the gel.
10. Repeat with three more pieces of filter paper.
11. Holding the stack, drain off all excess buffer from the plate. Wipe away
any droplets around the edge of the stack.
Semidry Electroblotter 2-5Thermo Scientific
Section 2
Setting Up
Gel Sandwich
Assembly (continued)
Anode
Membrane
Gel
Cathode
Transfer
Filter
Paper
Filter
Paper
Figure 2-1. Assembly Order

Semidry Electroblotter 3-1Thermo Scientific
Section 3 Using the System
1. Place and loosely tighten down the lid with the supplied black knobs.
The weight of the lid is usually enough, and the screws are not
required. Note: Do Not Use the screws for gels thicker than 1.5mm, or
when using more than 6 filter pads (total). If the screws are used, tip
the unit on an angle, to drain off any excess buffer that may have
squeezed out. This will safely remain in the moat around the electrode
plate.
2. Attach the power leads (red to red, black to black) to an appropriate
power supply. The red lead has a shroud, that will stop it from
attaching to the black cathode.
3. Run the blot.
Blotting takes place at a given migration rate for a specified time. The
units are mA times hrs. If you need to slow the transfer down, to say
coincide with the setting up of a probe, simply decrease the current (mA)
to match the added time you require.
(mA)(hr) Std setting = (mA)(hr) New setting
Alternatively, the current can be increased to decrease the time. This
assumes that you have determined an initial mAh value that works well for
the molecules you are interested in.
A current to use for a 45 min time period is based on the area (cm.cm) of
your gel (which is the resistor in this system). A range of 0.8 to 2 mA per
square centimeter of gel.
For example, if you had a 10 x 10cm gel, the area would be 100cm2, so the
current range would be 80mA (0.8mAcm-2x 100cm2) to 200 mA
(2mAcm-1x 100cm2).
Blots may also be run at constant voltage. Some power supplies have
difficulty sustaining steady voltages at these low voltage settings. If you
find that voltages are fluctuating, or that the power supply shuts itself off
when set on constant voltage, use constant current settings instead.
Running the Blot
Transfer settings

3-2 Semidry Electroblotter Thermo Scientific
Section 3
Using the System
Transfer Settings
(continued)
Read your power supply's instructions to ensure that the power supply will
work at a voltage lower than lOV. These voltages often occur in semidry
blotting. Contact the manufacturer regarding the unit's performance under
high current, low voltage conditions if you have any questions.
While general conditions can be described which will result in successful
transfer of most molecules, it should be noted that optimal transfer
conditions will vary based on the characteristics of the molecule you are
working with. Some factors that affect transfer rate and efficiency include
molecule size, charge, gel thickness and percentage, and hydrophobicity.
The reference list at the end of this manual provides useful information
that can help you choose optimal conditions for efficient transfer of a
specific molecule.
Factors That Affect
Transfer Efficiency
Protein
HEP-3/ Sequencing
gel - see HEP-1 set-
tings for other size
gels on this unit
DNA/RNA
Filter Paper
FP-1, 20 x 20cm, FP-6,
9 x 9cm, FP-4, 10 x
10cm, FP-7, 12 x 16cm,
or FP-5, 18 x 20cm
FP-2, 35 x 45cm, or
FP-3, 46 x 57cm
FP-1, FP-4, FP-5, FP-6, or
FP-7
Membrane
Nitrocellulose .45 or
0.2μ, PVDF 0.45μ or
0.2μ
Nylon Nylon
Transfer Buffer
Towbin Buffer, Buffer
Electroblot Buffer Kit,
ER-35, 3 Buffer System
Bjerrum and Schafer-
Nielsen
0.5X-1X TBE 0.5X-1XTBE, TAE, NAQ
Power Supply OSP-135 or equivalent High current/low volt-
age power supply OSP-135 or equivalent
Power Settings
Constant current 0.8-
3mA per cm2 gel sur-
face area 10-14 Volts
maximum
For entire gel, 1200-
1400 mA approximate-
ly 0.8mA/cm2/
produces ~5-8V
Constant Current 0.5-
3mA/cm2 gel surface
area 10-14 Volts maxi-
mum
Running Time
30 minutes to 2 hours
Needs to be experi-
mentally determined
(large molecules need
longer transfer times)
15-20 minutes at this
setting
30 minutes to 2 hours,
generally in the lower
range
Running Conditions

Semidry Electroblotter 4-1Thermo Scientific
Section 4 Troubleshooting
Problem Cause Solution
Transfer efficiency is poor • Current is too low
• Semidry transfer should be performed at constant
current. Current density should be between 0.5 and
3mA/cm2 of stack surface area.
• Power supply is inappropriate for semidry
transfer
• Many power supplies will shut off or blow a fuse
when run at the conditions required for semidry
transfer. Semidry transfer requires low voltage (often
less than 10V) and high current. Check with the man-
ufacturer of the power supply to determine whether
it is appropriate for semidry transfer.
• Transfer performed for too short a time • Increase the amount of time for transfer.
• Transfer sandwich was assembled in the
wrong order
• The semi-dry electroblotter is configured with the
cathode on the bottom, and anode on top. This
means that an upward transfer is being performed
rather than downward. Follow the instructions care-
fully when assembling the transfer sandwich.
• The pH of the transfer buffer is too close
to the isoelectric point of the protein • Try a more acidic or basic transfer buffer.
• Too much methanol in the transfer buffer
• Reducing methanol can help elute proteins from
the gel, but can reduce binding to nitrocellulose
membranes.
• High percentage gels restrict transfer
• Higher percentage acrylamide or crosslinker can
restrict elution of proteins. Use the lowest percent-
age acrylamide possible to separate your proteins.
• Puddles of buffer were present on the
cathode allowing the current to bypass the
stack
• Always clean up the lower plate before closing
the lid of the transfer apparatus. Do not squeeze the
stack excessively as this also creates puddles that
the current can pass through.
• The filter paper was too dry • Filter paper should be saturated with transfer
buffer before adding them to the sandwich.

4-2 Semidry Electroblotter Thermo Scientific
Section 4
Troubleshooting
Problem Cause Solution
Nitrocellulose membranes
Insufficient binding of
proteins to the mem-
brane
• Over-transfer through the
membrane
• Use 0.2micron pore size nitrocellulose instead
of 0.45micron, or use PVOF with a higher binding
capacity.
• Not enough methanol in the
transfer buffer
• Nitrocellulose binds proteins best when 20%
methanol is used in the transfer buffer.
• Low MW proteins are not
binding well or are being washed
away
• Use glutaraldehyde to crosslink the proteins to
the membrane and use Tween-20 in the wash
steps.
• SOS is preventing binding • Eliminate SOS in the transfer buffer.
PVDF
Smeared or swirled
transfer and missing
bands
• Membrane was dried out before
it was added to the transfer sand-
wich
• Membrane should be completely gray and
slightly translucent when added to the sandwich.
If it has dried out, rewet in methanol and equili-
brate in transfer buffer.
• Alcohol was not used to prewet
the membrane
• PVDF is hydrophobic and requires a short soak
in methanol prior to transfer.
• Air spaces are interfering with
contact between the gel and the
membrane
• Roll a test tube or pipet over the membrane
(make sure it is clean) before putting the rest of
the filter paper on the sandwich. Transfer will not
occur where the gel is not in contact with the
membrane.
• Electrophoretic conditions were
incorrect or not ideal
• Running conditions, sample preparation, per-
centage acrylamide, and many other variables
can affect the migration and resolution of pro-
teins. Review your electrophoresis conditions.
• Transferring at too high a current • Refer to the Running Conditions table.
• Membrane was not thoroughly
wetted.
Always pre-wet the membrane according to the
manufacturer’s instructions. White spots indicate
the dry areas of the membrane.
• Too much current
• Running at constant voltage can cause power
fluctuations that will cause overheating. A buffer
that has not been made correctly or that has too
high in ionic strength can also burn a gel by over-
heating. A cracked and dry gel often is an indica-
tor of overheating.

Semidry Electroblotter 5-1Thermo Scientific
Section 5 Technical Tips
How long will it take to blot the proteins from my gel?
Transfer times have to be determined experimentally. This is because
transfer time is dependent upon:
• Percentage of gel
• Type and amount of cross linking in the gel
• Type of protein: cytoplasmic, membrane, nuclear
• Size of protein
There is no formula for determining transfer time. There are too many
variables involved to give specific transfer conditions that will work for
every protocol.
Guidelines are:
• HEP-l: 2mA/cm2 of gel for 1 hour
These guidelines are just a starting point and exact conditions have to be
determined.
Different kinds of blotting
Western Blotting is a blotting method for proteins that use specific
antibodies attached to particular protein to help identify it. It is often
performed after SDS-PAGE or some other form of polyacrylamide gel
electrophoresis.
Southern Blotting is a method sometiems called hybridization because a
radioactive probe is "hybridized" or attached to specific pieces of DNA.
Northern Blotting is a similar method but the molecules involved are
RNA.
Both Southern and Northern blotting generally require the DNA or RNA
to first be separated out on an agarose gel.