Labnet ENDURO VE10 User manual

Copyright 2017 Labnet International, Version # 2
ENDURO™ VE10 Vertical Gel Electrophoresis Systems
User Manual
ENDURO VE10 System
E2010-PA
ENDURO VE10 Electroblotting System
E2010-PBA
Lit #M00608

Table of Contents
Page
Safety Precautions 1
Packing Lists 3
Care and Maintenance 5
Setting Up 6
Gel Casting 8
Gel Preparation 8
Gel Selection 9
Gel Pouring 10
Sample Preparation and Loading 11
Gel Running 12
Blotting Insert Setup 14
Blot Running 15
Appendix 20
Warranty 23
AccessoriesList 24

1
SAFETY PRECAUTION
WHEN USED CORRECTLY, THESE UNITS POSE NO HEALTH RISK.
HOWEVER, THESE UNITS CAN DELIVER DANGEROUS LEVELS OF ELECTRICITY AND ARE TO
BE OPERATED ONLY BY QUALIFIED PERSONNEL FOLLOWING THE GUIDELINES LAID OUT IN
THIS INSTRUCTION MANUAL.
ANYONE INTENDING TO USE THIS EQUIPMENT SHOULD READ THE COMPLETE MANUAL
THOROUGHLY.
The current to the unit, provided from external power supply, enters through the lid assembly,
providing a safety interlock to the user. When the lid is removed, the current to the unit is broken. DO
NOT attempt to use the unit without the safety lid correctly positioned. Always turn the power supply
off prior to removing the lid.
THE UNIT SHOULD NOT BE USED IF THERE IS ANY SIGN OF DAMAGE TO THE EXTERNAL
TANK OR LID.
ACRYLAMIDE IS A POWERFUL NEUROTOXIN IN SOLUTION FORM.
POLYMERIZED GELS CAN CONTAIN SOME UNPOLYMERIZED SOLUTION AND PROTECTIVE
GLOVES, SAFETY GLASSES AND CLOTHING MUST BE WORN.
THESE UNITS COMPLY WITH THE STATUTORY CE SAFETY DIRECTIVES:
73/23/EEC: LOW VOLTAGE DIRECTIVE: IEC 1010-1:1990 plus AMENDMENT 1:1992
EN 61010-1:1993/BS EN 61010-1:1993

2
Important Notice
Care and Maintenance:
These products and associated accessories should never come into
contact with the following cleaning agents, as these will cause
irreversible and accumulative damage:
Acetone
Chloroform
Methanol
Carbon tetrachloride
Phenol
Alkalis
Ethanol
Isopropyl alcohol
Water at temperatures above 60°C will cause damage to the acrylic
tanks, trays and other parts.
The tanks should be thoroughly rinsed with warm or distilled water but vigorous
cleaning is not necessary or advised. Air drying is recommended before use.
Cleaning Products and Accessories
Products are best cleaned using warm water and a mild detergent.
The units should only be cleaned with the following:
Warm water with a low concentration of soap or other compatible mild detergent.
Compatible detergents include dishwashing liquid, Hexane and Aliphatic
hydrocarbons.
The units should not be left in detergents for more than 30 minutes

3
PACKING LISTS:
ENDURO VE10 System
Units include tank, lid, internal module and electrodes and include the following
accessories:
Glas
s Plates
Combs
Casting
Base
Cooling
Pack
Cables
E2010
-
PA
VE10 Vertical
Gel System
(E2110-NG-2) Glass
Plates, Notched, Pk/2
(E2110-PG-1-BS) Glass
Plates, Plain with bonded
1mm spacers, Pk/2
(E2110-DP) Dummy Plate
(E2110-12-1)
qty of 2, 1mm
thick, 12 sample
combs
E2110-PC E2110-CP E1107-EP
ENDURO VE10 Electroblotting System
Units include tank, lid, internal module, electrodes, Blotting module and include the
following accessories:
Glass Plates
Combs
Casting
Base
Cooling
Pack
Cables
ENDURO VE10
Electroblotting
System
(E2110-NG-2) Glass
Plates, Notched, Pk/2
(E2110-PG-1-BS) Glass
Plates, Plain with
bonded 1mm spacers,
Pk/2
(E2110-DP) Dummy
Plate
(E2110-12-1)
qty of 2, 1mm
thick, 12 sample
combs
E2110-PC E2110-CP E1107-EP
Electroblotting
module
(E2110-B-MC) 4 Blotting Cassettes, (E2110-B-FP) Pack of 8 Fiber pads

4
The packing lists should be referred to as soon as the units are received to
ensure that all components have been included. The unit should be checked
for damage when received. If damaged, contact carrier and save the box for
inspection. Please contact Labnet International if there are any problems or
missing items.
Usage Guidance and restrictions:
• Maximum altitude 2,000m.
• Temperature range between 4°C and 60°C.
• Maximum relative humidity 80% for temperatures up to 31°C decreasing linearly to
50% relative humidity at 40°C.
• Not for outdoor use.
This apparatus is rated POLLUTION DEGREE 2 in accordance with IEC 664.
POLLUTION DEGREE 2, states that: “Normally only non-conductive pollution
occurs. Occasionally, however, a temporary conductivity caused by condensation
must be expected.”
Care and Maintenance:
Cleaning ENDURO VE10 System
Units are best cleaned using warm water and a mild detergent. Water at
temperatures above 60°C can cause damage to the unit and components. The
tank should be thoroughly rinsed with warm water and distilled water to prevent build
up of salts but care should be taken not to damage the enclosed electrode.
Vigorous cleaning is not necessary or advised. Air drying is recommended before
use.
The units should never come into contact with the following cleaning agents,
these will cause irreversible and cumulative damage:-

5
Acetone, Phenol, Chloroform, Carbon tetrachloride, Methanol, Ethanol, Isopropyl
alcohol, Alkalis.
RNase Decontamination
This can be performed using the following protocol:-
Clean the units with a mild detergent as described above.
Wash with 3% hydrogen peroxide (H2O2) for 10 minutes.
Rinse with 0.1% DEPC- (diethyl pyrocarbonate) treated distilled water,
Caution: DEPC is a suspected carcinogen. Always wear gloves and safety
glasses.
RNaseZAP™ (Ambion) can also be used. Please consult the instructions for use
with acrylic gel tanks.
Questions and Service:
Should you have a question about the operation of the Spectrafuge Mini Centrifuge or
if service is required, contact Corning at: 800-492-1110. Do not send in a unit for
service without first calling to obtain a repair authorization number. Should the unit
require return to Corning for service, it should be properly packed to avoid damage.
Any damage resulting from improper packaging shall be the responsibility of the
user.

6
Setting up the ENDURO VE10 Gel Tanks:
Instructions for installing Electrical Leads.
1. Note the position of the lid on the unit. This shows the correct polarity and the
correct orientation of the cables, black is negative and red positive.
2. Remove the lid from the unit. Note if the lid is not removed, fitting the cables may
result in un-tightening of the gold plug and damage to the electrode.
3. Screw the cables into the tapped holes in the lid as fully as possible so that there
is no gap between the lid and the leading edge of the cable fitting.
4. Refit the lid.
The unit is now ready for use.
Vertical Gel Casting Using the ENDURO VE10 Gel Casting System:-
1. Clean a set of glass plates for each gel first with distilled water and then with
70% ethanol. One set of glass plates constitutes one notched glass plate and
one plain glass plate with bonded spacers. When using a triple glass plate
sandwich, two notched glass plates, one set of free spacers and a set of plain
glass plates with bonded spacer. The plain glass plate is positioned outermost,
then a notched glass plate, free spacers and second notched glass plate.
Alternatively, accessory notched glass plates with bonded spacers are available.
All glass plates, modules and casting base accessories must be completely
dry for setup. Wet components are more likely to misalign and cause leaks.
2. Assemble the glass plates so that the bottom of the glass plates and the spacers
are perfectly aligned. For triple plate sandwiches, the free spacers need to be
perfectly aligned which is best performed using a small spacer or comb to push
the spacers apart. Notched glass plates with bonded spacers do not need

7
manual alignment. NOTE: The spacers bonded to the glass plates are
slightly longer than the plates and marked with an arrow to indicate the top.
3. The ENDURO VE10 Gel insert contains pressure bars which impart even
pressure onto the edge of the glass plate. Ensure that the pressure bars are
adequately open for the thickness of spacer used. The bar can be opened by
sliding open clamps. When using a triple glass plate sandwich, the pressure bars
will need to be in the completely open position.
4. Position the ENDURO VE10 Gel module on a flat surface. Do not insert the
ENDURO VE10 Gel module into the casting base at this point.
5. Place the glass plate/spacer assembly into the Vertical Gel Insert between the
pressure bar and the blue gasket. Check that the bottoms of the glass plates are
touching the bench and fully tighten the sliding clamps. When only one gel is
being run, the dummy plate must be used in the second position and fully
tightened. NOTE: Be sure that the glass plates and the bottom of the
spacers are evenly aligned.

8
6. Open the cam handles on the casting stand and position the cams so they face
downwards. Position the ENDURO VE10 Gel insert on the gasket of the casting
stand so the key holes are facing the cams. The top of the ENDURO VE10 Gel
insert will need to be pushed down very slightly to insert the cams.
7. With the cam handles facing directly downwards, rotate the cams in the opposite
direction 1800 or until the insert has tightened down onto the gasket. Do not
overturn as this will cause the glass plates to push upwards and the
assembly will be more likely to leak.
NOTE: Always reverse the gasket after casting to avoid indentations. Do not
leave glass plates tightened into the casting stand for long periods of time as
this can cause damage the silicone mat. The unit is now ready for gel
preparation.

9
Vertical Gel Casting:
A) Screw Version B) Sliding clamp version
Gel Preparation:-
1. Stock solutions for SDS PAGE gels should be prepared prior to use and chilled.
For native gel formulas and running conditions, please consult the appendix. The
protocol below is given for use of the standard stock solutions.
2. Table 1 below shows the total volume of gel solution required. In subsequent
tables, amounts of gel and solutions are given for two 1mm thick gels. If gels of
other thickness are to be run adjustments will need to be made.
1) Put together bonded
spacer plain glass plate
with notched plate
3A) Fully tighten
screws ensuring
not to wobble unit.
2)
Insert
inside
pressure
bar with notched plate
innermost touching the
gasket and module on a
flat surface away from the
casting base
3B) Fully slide
clamps tight
ensuring not to
wobble unit.
4) Insert into casting
base. Push the cams
into the holes in the
insert. Turn cams
about 900or until
tight. Do not over
tighten.
5) Pour resolving gel
and allow to set.
Then stacking gel
solution and insert
comb.
6) Once set, transfer
to tank and fill inner
and outer chambers
with buffer.

10
Table 1.
ENDURO VE10
Total Gel volume for a 1mm thick gel.
For different thicknesses of gel, multiple the below amounts by the spacer
thickness.
Single – one gel, one dummy plate 7.5 mL
Double – two gels 15 mL
Using a Triple Plate sandwich – 4 gels 30 mL
Gel Selection:
Care should be taken when selecting the pore size of the gel to be used. These
formulas are for Tris- glycine-SDS gels
The pore size or % of gel determines the resolving ability given different sizes of
protein. See Table 2 below which details which percentage of gel to use to separate
the sizes of proteins indicated.
Table 2.
Acrylamide Percentage
Separating Resolution
5 % 60 - 220 KD
7.5 % 45 - 120 KD
10 % 25 - 75 KD
12% 14.4 – 65 KD
15 % 6.5 -45 KD
17.5% 5.5 – 30 KD
3. Using the stock solution provided in the appendix prepare gel solutions as per
tables below. First mix the ddi Water, 30% Acrylamide solution and the 4x TRIS-
SDS solutions. After mixing, degas for 5 minutes to remove free oxygen (which
will inhibit polymerization).
4. To the above solution add the ammonium persulfate and TEMED and mix gently
to avoid air bubbles.

11
Table 3: Preparation of the resolving gel solution for two 10 x 10cm gels using
1 mm spacers.
Solution 5% 7.50% 10% 12% 15% 17.5%
Distilled Water 8.7 mL 7. 5mL 6.3 mL 5.25 mL 3.75mL 2.5 mL
30 % Stock
Acrylamide
Solution 2.5 mL 3.75 mL 5 mL 6 mL 7.5 mL 8.75mL
4 X Tris-SDS
Solution pH 8.8 3.75 mL 3.75 mL 3.75 mL 3.75 mL 3.75 mL 3.75mL
10 %
Ammonium
Persulphate 150 µl 150 µl 150 µl 150 µl 150 µl 150 µl
Gel Pouring:
For discontinuous gels:-
1. Insert the comb between the glass plates and mark a point on the glass plates
1cm below the bottom of the teeth. This is the level for the resolving gel.
2. Add 15 µl of TEMED to the resolving gel solution.
3. Fill the glass plates to the line and avoid generating any air bubbles. Filling must
be performed quickly before the TEMED causes the gel to become too viscous.
4. Carefully, overlay the gel with 1 ml of 1% Isobutanol, Isopropanol or distilled
water. When using distilled water, extra care must be taken to ensure there is no
mixing with the gel solution.
5. Allow the resolving gel to polymerize. This usually takes 15 to 30 minutes, but
can vary due to freshness of reagents used. If polymerization time is excessive,
use a fresh stock solution or add more APS and TEMED.
6. Prepare the stacking gel using Table 5 below as a guide. (See appendix for
stock solutions.)

12
Table 5.
Solution
ENDURO VE10
Distilled Water 4.2 mL
30 % Stock Acrylamide
Solution 0.65 mL
4 X Stacking Gel Tris-
SDS Solution pH 6.6 1.6 mL
10 % Ammonium
Persulphate 67µl
7. Carefully mix the stacking gel solution, avoiding generating air bubbles.
8. Pour off the overlay liquid and rinse the gel with distilled water.
9. Add 6.7µl of TEMED to the stacking gel solution. Mix well. Use a Pasteur pipette
to fill the glass plates up to the top with stacking gel solution.
10.Carefully insert the comb making sure that no air bubbles are trapped under the
ends teeth as these will inhibit sample progression.
11.Allow the stacking gel polymerize for 30 minutes.
For continuous gels:
4. Follow the instructions for mixing the acrylamide solution. Add 15µ of TEMED
and mix well but avoid generating air bubbles.
5. Fill the glass plates to 1 cm below top of notched plate, again avoiding
generating any air bubbles. Filling must be performed quickly before the TEMED
causes the gel to become too viscous.
6. Carefully insert the comb making sure that no air bubbles are trapped under the
teeth as these will inhibit sample progression.
7. Let the gel polymerize. Usually this takes around 15 minutes but this can vary
due to the freshness of the reagents used. If polymerization is taken a lot longer
than this, use fresher stock solutions or add more APS and TEMED.

13
Preparation of denatured protein samples for loading:
The instructions given below are for denatured samples. For Native samples, please
consult a laboratory handbook.
1. Prepare the protein samples for loading. The volume of sample depends on
the capacity of the wells. See well chart in the Appendix.
2. Using a 0.5 ml micro-centrifuge tube or other convenient receptacle, combine
the protein sample and 4 X sample buffer. It is always advisable to use
protein markers in one of the end lanes to indicate sizes of bands. These
should be prepared according to the manufacturer’s instructions.
3. Heat the samples in a water bath or heating block for 2 minutes to denature.
NOTE: heating should be done under a fume hood.
4. Centrifuge the samples in a microcentrifuge for 20 seconds at 12,000 rpm.
The protein samples are now ready to load.
Loading the samples:
1. If desired, fit the previously frozen cooling pack(s) into the tank. The longest side
of the ice pack should be positioned horizontal with the side(s) of the tank and
pressed into the recess. One pack is supplied as standard. Additional packs can
be purchased.
NEVER FIT THESE UNDERNEATH THE MODULE IN THE BOTTOM OF
THE TANK AS THIS WILL PREVENT THE FLOW OF CURRENT
THROUGH THE GEL AND CAUSE SLOW RUNS AND OVER-HEATING.
2. Remove the combs with a gentle rocking motion; rinse the well out with ddi H2O
to eliminate any residue.
3. Transfer the Inner gel module containing cast gels into the main tank in the
correct orientation as indicated +ve on the module aligned with +ve on the tank,
-ve on the module aligned with -ve on the tank.
4. Fill the outer tank with 1 x reservoir buffer. See Appendix for recommended
running buffer solution. Table 6. Shows the volume of buffer required.

14
Table 6.
Buffer Volume
ENDURO VE10
Minimum
–
Inner tank is filled to above the wells.
Outer Tank is filled to just flood the bottom of the glass
plates. Cooling potential is at a minimum which may
affect resolution.
250 mL
Maximum
–
Inner tank is filled to above the wells.
Outer Tank is filled to the maximum fill line. Cooling is
high offering good resolution of samples.
1200 mL
Using the cooling packs
–
Inner tank is filled to
above the wells. Cooling packs are inserted behind the
gels. Outer Tank is filled to the maximum fill line.
Cooling is at a maximum. Only 1 cooling pack comes
standard with unit.
1000 mL
5. Load the samples into the wells using a pipette tip taking care not to damage the
wells or induce any air bubbles.
6. Fill any unused wells with 1X sample buffer.
7. It is a good idea to note the orientation and order the samples were loaded in.
This can be done by noting which samples were loaded adjacent to each
electrode.
Gel Running:
1. Fit the lid and connect to a power supply.
2. Consult Table 7 for details on recommended power supply voltage settings.
3. Run times vary with concentration and protein size. When the dye front is
approximately 1 cm, from bottom, turn off the power supply. If resolving proteins
less than 4Kd, the power supply should be turned off sooner.
4. Always unplug the power cord from the power supply prior to removing the lid.
5. Remove the gel running module, first emptying the inner buffer into the main
tank. Buffer can be re-used but this may affect run quality.

15
6. Release the pressure bars and gently pry apart the glass plates. The gel will
usually stick to one of the plates and can be removed by first soaking in running
buffer and then gently lifting with a spatula.
7. The gel is now ready to be stained with Coomassie or silver stain for specific
band identification and further analysis. If using the Enduro GDS for imaging you
will need the white light conversion screen.
Table 7.
Recommended Voltage
and Current
settings
for 1mm thick, 12% gels.
VE10
One gel 90-225V,
20-45mA
Two gels 90-225V,
40-90mA
Three gels 90-225V,
60-135mA
Four gels 90-225V,
80-180mA
The recommended power condition for optimal resolution with minimal thermal band
distortion is 150 volts, constant voltage setting. No adjustment of the setting is
necessary for thickness or number of gels. The usual run time is approximately 40-
45 minutes. Current should be approximately 50mA per gel (120mA for two gels) at
the beginning of the run. During the 45 minute run, the current will slowly drop to
about 20mA per gel. This drop is caused by the change in buffer ions in the gel,
causing a slow rise in the resistance in the gel. As one would expect from the Ohms
law (V=I*R), at constant voltage (V) a rise in the resistance (R) results in a drop in
the current (I).

16
Protein Blotting using the ENDURO VE10 Electroblotting Insert
Setting up the cassette sandwich: (Common buffer solutions are listed in the
Appendix.)
1. Each blot sandwich should be set up as follows:-
a. Cassette clamp -ve (black) side placed in a tray or other suitable surface.
b. Pre-soaked fiber pad.
c. Two pieces of .45µm filter paper, pre-soaked in buffer.
d. Gel - cut the left hand corner for indexing,
e. Transfer membrane. Follow manufacturer’s directions for presoaking.
Smooth out any air bubbles that may be trapped under the membrane.
f. Two pieces of filter paper, pre-soaked in buffer.
g. Pre-soaked fiber pad.
h. Cassette clamp +ve (red) side slotted into the groove in the bottom of the
black cassette.

17
Note: do not handle the membrane without gloves.
2. Assemble the fiber pads, filter papers, gel and transfer membrane in the above
order and roll with a pipette to remove any trapped air. Place on the black and
red cassette with the membrane facing the anode (red) side, close the hinge
carefully so as to not disturb the sandwich.
3. Fill the tank with buffer solution up to the maximum fill line indicated on the side
of each unit. See the Appendix for recommended buffer solutions. Improved
transfer can be obtained by using chilled buffer.
Table 8. shows the volume of buffer required.
Buffer Volume
ENDURO
Electroblotting
One Cassette 1380 mL
Two Cassettes 1290 mL
Three Cassettes 1200 mL
Each cooling pack takes the place of 100 mL of buffer.
Blot Running Conditions:
1. Insert the cassettes into the slots in the module with the black side of each
adjacent to the negative electrode. It is a good idea to note the orientation and
order in which the blot sandwiches were loaded.
2. Use of a magnetic stirring bar and plate is recommended to mix the buffer to give
consistency of transfer. A 4mm diameter stirring bar should be placed
underneath the module, in the center of the tank. The cooling pack provided, pre-
frozen, can be inserted at the side or front of the tank for extended blots.
Additional cooling packs can be purchased as accessories to further aid cooling.
3. Insert the module, attach the lid and connect to a power supply.
4. Consult Table 9 for details on recommended power supply voltage settings and
blot times. Please note voltages and current will vary according to the amount of
cassettes, type and temperature of buffer and thickness and percentage of gel.
This will also affect quality of transfer so adjust the time of the blot to your
particular samples and conditions.

18
5. When the blot time is completed, turn the power supply off and remove the lid of
the unit.
6. Remove the cassettes from the main tank.
7. Lift the hinge of each cassette and gently pry apart the blot sandwich and remove
the membrane from the gel.
8. The membrane can now be further processed. Remember to save the filter paper
behind the gel to check for blow through of smaller proteins.
Table 9. Recommended voltage and current settings.
Duration of Blot
Volts
/
mAmp
One Hours 100V, 400mA
Three Hours 50V, 200mA