Cup Loading Tray for the PROTEAN IEF Cell ®

Cup Loading Tray for the PROTEAN IEF Cell ®
Cup Loading Tray for the
®
PROTEAN IEF Cell
For technical service
call your local Bio-Rad office or
in the U.S. call 1-800-4BIORAD
(1-800-424-6723)
On the Web at discover.bio-rad.com
Table of Contents
Section 1
1.1
1.2
1.3
1.4
Section 2
2.1
2.2
2.3
2.4
2.5
2.6
2.7
Section 3
3.1
3.2
3.3
Section 4
Section 5
Section 6
Introduction ....................................................................................1
Overview..........................................................................................1
Introduction ....................................................................................1
Description ......................................................................................3
Specifications ..................................................................................4
Instructions ...................................................................................... 4
General Overview ............................................................................4
IPG Strips Rehydration Solution Preparation and
Strip Rehydration ............................................................................5
Transfer and Placement of Rehydrated IPG Strips in the
Focusing Tray ..................................................................................5
Placement of Movable Electrodes....................................................6
Positioning Sample Loading Cups and Loading Sample..................6
Focusing Conditions ........................................................................8
After Focusing Run is Complete ......................................................9
Cleaning, Maintenance, and Chemical Compatibility ...................... 10
Focusing Tray ................................................................................10
Movable Electrode Assemblies ......................................................11
Cups ............................................................................................11
General Guidelines for Sample Preparation .................................... 11
Troubleshooting ............................................................................ 12
Warranty ........................................................................................13
Section 1
Introduction
1.1
Overview
The Cup Loading Tray for the PROTEAN IEF cell is designed for the optimal resolution and
separation of proteins with isoelectric points (pI) at either extreme of the pH range. The Cup
Loading Tray can accommodate up to 12 IPG strips from 7 cm to 24 cm in length.
The Cup Loading Tray includes a tray base, a pair of movable electrode assemblies, and one bag
each (120 count) of 100 and 150 µl sample cups. No modifications to the PROTEAN IEF cell are
needed for compatibility with the Cup Loading Tray.
The Cup Loading Tray offers the following benefits:
•
Enhanced resolution and separation of proteins with pIs at extreme pH ranges
•
Optimal focusing for users of ReadyStrip™ IPG strips pH 3-6, pH 7-10, and Micro-range
ReadyStrip IPG strips pH 6.3–8.3
•
Tray runs up to 12 IPG strips per run for high throughput
•
Tray accommodates 7 cm to 24 cm IPG strips with movable electrode assemblies
•
Two sample cup sizes, 100 and 150 µl, are available to allow greater flexibility
•
The Cup Loading Tray is compatible with all PROTEAN IEF cells, so no modification to
existing PROTEAN IEF cell is necessary.
1.2
Introduction
Isoelectric focusing of proteins in the acidic and basic pH ranges is often associated with
streaking due, in part, to protein precipitation when utilizing standard in-gel rehydration of IPG
strips. Protein resolution can be markedly improved when the proteins are applied to the IPG
strips by means of a sample cup positioned in a discreet area of a rehydrated IPG strip.
The Cup Loading Tray allows for sample application via cup loading after rehydration of the IPG
strips. Certain conditions make cup loading the preferred method of sample loading. The
examples below compare in gel rehydration resolution with cup loading resolution of an E. coli
sample on pH 3-6 and pH 7-10 IPG strips.
1
pH 3
pH 6
pH 3
pH 6
10%
20%
A. pH 3-6 cup loading tray
pH 7
B. pH 3-6 in-gel rehydration
pH 10
pH 7
pH 10
8%
16%
C. pH 7-10 cup loading tray
D. pH 7-10 in-gel rehydration
Figure 1.
In-gel rehydration: 400 µg E.coli lysate in 8M Urea, 4% Chaps, 50 mM DTT, 0.2% Bio-Lytes.
Cup loading: 400 µg E.coli lysate in 100 µl 8M Urea, 4% Chaps, 50 mM DTT, 0.2% Bio-Lytes.
A. pH 3-6 cup loading at cathode (-), B. pH 3-6 in-gel rehydration
C. pH 7-10 cup loading at anode (+), D. pH 7-10 in-gel rehydration
2
1.3
Description
The Cup Loading Tray for use with the PROTEAN IEF cell consists of the following components:
Electrode
Spring cup
Large (150 µl) sample cup
Small (100 µl) sample cup
Spring clip
µl
Right handle
Electrode
Electrode
contact area
Left handle
Cup loading tray
Electrode
contact area
Tray base
Figure 2. Diagram of Cup Loading Tray components.
1. A twelve-cup strip that holds up to 100 µl of sample per cup and a twelve-cup strip that holds
up to 150 µl of sample per cup.
Note: For precise mechanical fit, a minimum of two cups is required. A single sample cup
provides an incomplete “stop” on the channel walls and the gel could be punctured.
2. A twelve channel focusing tray that accommodates IPG strips from 7cm to 24 cm.
3. Two movable electrode assemblies, each having 12 individual teeth with cavities which allow
mineral oil to flow through the entire channel of the focusing tray.
3
Teeth
Cavities
Figure 3. Movable electrode assemblies.
1.4
Specifications
Material
IPG strip size capacity
Tray Capacity
Electrodes
Inter-electrode distance
IPG strip rehydration
Polycarbonate
7 cm–24 cm
Up to 12 IPG strips per run
Platinum wire on movable electrode assembly
User defined. Dependent on IPG strip length
Rehydration/equilibration tray specific to IPG strip length
Section 2
Instructions
2.1
General Overview
Isoelectric focusing with the Cup Loading Tray includes the following steps:
1. Prepare rehydration solutions and rehydrate IPG strips overnight in rehydration tray with
appropriate volume of rehydration buffer.
Note: The Cup Loading Tray is not intended for passive or active IPG strip rehydration. Please
use appropriate rehydration/equilibration tray.
2. Prepare sample.
3. Transfer the rehydrated IPG strips from the rehydration tray and place the strips gel side up
into the focusing tray’s channels.
Note: Reduced rehydration times result in incomplete rehydration of the IPG Strips causing an
imperfect seal between the sample cup and the IPG strip.
4. Optional. Place electrode wicks at anode and cathode.
5. Position the movable electrode assemblies on top of the strips at the anode and cathode
ends. Ensure the spring clips are in contact with the electrode contact areas.
6. Place the sample loading cups on the rehydrated IPG strips near the anode for basic pH
range and near the cathode for acidic pH range IPG strips.
7. Load sample in sample cup and overlay with mineral oil.
8. Overlay IPG strips in each channel with mineral oil.
9. Transfer tray to cooling platform in PROTEAN IEF cell and start isoelectric focusing.
4
2.2
IPG Strip Rehydration Solution Preparation and Strip Rehydration
1. Prepare rehydration sample buffer and rehydrate IPG strips just prior to sample cup-loading
and isoelectric focusing. Add the indicated volume of rehydration buffer to each of the
required channels in the rehydration tray to rehydrate to 0.53 mm thickness.
Note: Rehydration to 0.53 mm thickness ensures a complete seal between the cup and the
IPG strip. The following volumes are 10% greater than those recommended in the ReadyStrip
IPG Strip instruction manual.
IPG Length
7 cm
11 cm
17 cm
18 cm
24 cm
Rehydration
volume for
cuploading
135 µl
200 µl
330 µl
345 µl
450 µl
2. Carefully remove the IPG strip cover sheet and place the IPG strip gel side down in the
rehydration trays. To ensure even rehydration of the IPG strips, the entire surface to the IPG
strips must be completely wetted.
Figure 4. Removal of IPG strip cover sheet.
3. Overlay IPG strips with mineral oil and rehydrate strips ~ 12 hours (or overnight). To maintain a
constant temperature during rehydration, place the rehydration tray on the cooling platform of
the PROTEAN IEF cell and set-up the rehydration program. (See PROTEAN IEF cell manual
for details).
2.3
Transfer and Placement of Rehydrated IPG Strips in the Focusing Tray
1. Remove the rehydrated IPG strips from the rehydration tray. Remove excess mineral oil by
carefully blotting the IPG strip on filter paper wetted with DI water.
5
2. Place the rehydrated IPG strip, gel side up, in the focusing tray. Place the anode side (marked
with + on ReadyStrip IPG Strips) of the 17 cm, 18 cm, and 24 cm strips flush against the
anode (left) side of the focusing tray. Place the anode side of the 7 cm and 11 cm approximately 4 cm from the anode side of the tray to ensure proper placement and contact of the
cathode electrode assembly with the electrode contact area.
Movable
electrode
24 cm IPG strip
-
Movable
electrode
18 cm IPG strip
-
Movable
electrode
17 cm IPG strip
-
Movable
electrode
11 cm IPG strip
-
Movable
electrode
7 cm IPG strip
-
Movable
electrode
Tray well
17 cm IPG strip
-
A
A
Electrode
contact area
Electrode
contact area
Figure 5. Correct placement regions for various IPG strip lengths.
2.4
Placement of Movable Electrode Assemblies
1. Place a movable electrode assembly at the cathode and anode end of the IPG strip. Make
sure the electrodes make contact with the electrode contact areas on the front of the focusing
tray.
Note: If the electrode screws, indicated by the letter “A” in Figure 5, on the front of the focusing tray interfere with the placement of the moveable electrode assembly, reposition the IPG
strips until the electrode assembly clears the electrode screws.
Optional: Electrode wicks can be inserted at this point. Carefully place damp electrode wicks
on the gel surface prior to positioning the anode and cathode electrode assemblies.
2.5
Positioning Sample Loading Cups and Loading Sample
1. Determine which sample cup size will be used and cut the required number of sample cups
from a twelve-cup strip with scissors. A minimum of two sample cups is needed for a tight,
leak proof fit in the focusing tray.
6
Cut
Locking
tabs
Connecting bridge
Figure 6. Twelve-cup strip.
Note: When cutting the indent of the bridge between two cups, ensure that no burrs remain in
the cut that would prevent the bridge from sitting directly on the wall that divides two channels
in the focusing tray.
2. Place the sample cups on the gel surface near the anode for basic pH range IPG strips and
near the cathode for acidic pH range IPG strips. To position the sample cups properly, make
sure that the bridges connecting two cups are pushed down onto each wall that divides the
focusing tray channels.
Pipet tip
Cup
Sample
Tray well
IPG strip and gel
Tray
Figure 7. Side view of Focusing tray with IPG strips, cups, and sample.
Note: Improper placement of the sample cup can result in sample leaking.
3. When the cups are securely positioned, add the protein sample to the sample cups. We
recommend a maximum of 100 µl for the smaller cups and a maximum of 150 µl for the large
volume cups. We have observed improved resolution with smaller volumes containing a higher
protein concentration, and recommend using the maximum protein concentration possible in
which proteins do not precipitate.
7
Figure 8. Loading sample into cup with pipet.
Optional: The integrity of the seal between the gel surface and the cup can be verified by
adding some rehydration buffer to the cup prior to loading the sample. When a leak proof seal
is confirmed, carefully remove the rehydration buffer and replace with the sample.
4. Overlay each sample cup with mineral oil to prevent evaporation.
5. Completely overlay each strip with mineral oil.
6. Carefully place the focusing tray with the properly positioned electrodes and sample cups on
the temperature-controlling platform of the PROTEAN IEF cell.
Figure 9. Insertion of Focusing tray into PROTEAN IEF cell.
2.6
Focusing Conditions
Focusing conditions will vary with sample composition, sample complexity, IPG pH range and
strip length. The following parameters are recommended guidelines. We recommend maintaining
a current limit of 50 µA per strip to minimize excessive heat generation and subsequent increase
in the possibility of burned regions in the IPG strips.
8
Recommended Focusing Parameters
7 cm
11 cm
17 & 18 cm
24 cm
30 min.
30 min.
30 min.
30 min.
Step 2*:
4,000 V
8,000 V
10,000 V
Slow Voltage Ramping
60 min.
—
—
—
60 min.
—
—
—
60 min.
—
—
60 min.
Step 3*:
4,000 V
8,000 V
10,000 V
Rapid Voltage Ramping
8–10 kV hrs.
—
—
—
15–20 kV hrs.
—
—
—
30–40 kV hrs.
—
—
40–60 kV hrs.
Step 1: 250 V
Rapid Voltage Ramping
* The maximum attainable voltage is determined by the combined final conductivity of the IPG
strip, sample, and rehydration buffer. A lower final voltage does not adversely affect resolution
provided that the set volt-hours are reached.
2.7
After Focusing Run is Complete
1. Remove the focusing tray from the PROTEAN IEF cell when the focusing run is complete by
lifting the tray by the handle with your thumbs.
Figure 10. Removal of Focusing tray from PROTEAN IEF cell.
2. Remove the cups from the focusing tray by inserting the enclosed forceps underneath the
connecting bridge, or by pinching the wall of the cup with forceps and lifting upwards. If a full
strip of cups (12) is used, you may have to lift the cups in a few places along the strip.
9
Figure 11. Connecting bridge method.
Figure 12. Pinching cup walls method.
3. Remove the IPG strips from the focusing tray. Carefully blot the IPG strip on clean, wetted
filter paper to remove excess oil and sample buffer. Equilibrate the IPG strips as required prior
to the 2nd dimension. See the ReadyStrip IPG Strip instruction manual for details.
Section 3
Cleaning, Maintenance, and Chemical Compatibility
Proper cleaning and maintenance of the Cup Loading Tray components will ensure the best
results while providing the longest life for the product.
3.1
Focusing tray
The focusing tray is made of polycarbonate. Several chemicals are not compatible with
polycarbonate: acetone, acetonitrile, benzene, chloroform, hexane, hydrazine, methylene chloride,
and acetaldehyde. This list is representative, but not exhaustive. The IEF sample preparation
reagents at the concentrations normally used for IEF, such as ampholytes, various detergents,
and reducing agents, including tri-butyl phosphine, have no effect on the trays.
Clean the focusing tray after each use. Use the nylon cleaning brushes included with the
PROTEAN IEF cell or a soft-bristled brush with hot water and an Alconox® type of detergent
called Liqui-Nox®, which is specifically designed for removing greasy substances, to remove all
residual mineral oil and sample solution. Rinse with DI water and air dry. Do not use ethanol to
wash or dry the focusing tray. Use of ethanol will craze or crack the tray and voids the warranty.
Note: Do not try to remove the mineral oil with any solvent, as most solvents will likely damage
the focusing tray and void the warranty.
10
3.2
Movable Electrode Assemblies
Use the nylon cleaning brushes included with the PROTEAN IEF cell or a soft-bristled brush to
clean the movable electrode assemblies with hot water and Liqui-Nox. Take care to ensure the
platinum wire does not break. Rinse with DI water and air dry. Do not use ethanol to wash or dry
the movable electrode assemblies.
3.3
Cups
The Cup Loading Tray's cups are designed to be disposable and are not recommended for
reuse.
*Alconox and Liqui-Nox are registered trademarks of Alconox.
Section 4
General Guidelines for Sample Preparation
See ReadyStrip IPG Strip instruction manual for general guidelines for sample preparation. For
additional information on sample preparation, go to www.ProteomeWorksSystem.com
11
Section 5
Troubleshooting
Problem
Cause
Solution
1. Sample is
leaking from
the sample
cups.
Cup positioning is incorrect.
When positioning the cups, press down
on the connecting bridge until it stops
on the channel wall of the focusing tray.
(See illustration on page 7 .)
The cut edge from the
connecting bridge contains
a burr that prevents the
connecting bridge from
proper positioning on the
channel wall.
Make sure to use a sharp cutting
device such as scissors to evenly cut
the connecting bridge. Inspect cut to
ensure that it does not contain any
burrs.
The cup is positioned in an
IPG area that is not
completely rehydrated.
Make sure the IPG strips are rehydrated
evenly and thoroughly. See ReadyStrip
IPG Strip Manual for correct rehydration
directions. Make sure the correct
volume is used for the selected IPG
strip. See table in Section 2.2.
The cup itself is
malfunctioning.
Replace cup.
Incorrect positioning of the
electrode assemblies.
Make sure the electrodes are positioned
directly on the IPG strips. See page 6
for correct positioning.
2. Initial low or
zero current
(no electrical
contact)
When using electrode wicks make sure
they are wetted properly and are placed
on the IPG strips and the electrode
assemblies are in contact with the
electrode wicks.
Make sure the movable electrode
assemblies make contact with the
electrode contact area on the focusing
tray.
Platinum wire on electrode
holder has been
compromised.
12
Inspect the platinum electrode wire to
make sure it is intact. If in doubt, use a
voltmeter to check for continuity.
Problem
Cause
Solution
3. Sample wicks
out of sample
cups.
The sample cups are
disposable. When re-using
the sample cups, salts
can build up in the
corners and provide a
capillary path for the
sample to travel.
For optimum performance use new
sample cups for each focusing run.
4. Programmed
max. voltage is
not reached
with a current
limit of 50 µA
per strip.
Final combined conductivity
of the IPG strip, sample,
and rehydration buffer
determines the maximum
attainable voltage.
Run at maximum attainable voltage for
longer time to arrive at the required
number of volt-hours.
If possible, reduce the conductivity of
the rehydration buffer by reducing the
Bio-Lyte® concentration.
Increase the current limit per strip.
However, higher current limits generate
more heat and increase the possibility of
burned regions in the IPG strips.
Section 6
Warranty
The Cup Loading Tray is warranted for 1 year against defects in materials and workmanship. If
any defects should occur during this warranty period, Bio-Rad Laboratories will replace the
defective parts without charge. However, the following defects are specifically excluded:
•
•
•
•
•
•
Defects caused by improper operation
Repairs or modifications performed by anyone other than Bio-Rad Laboratories or their
authorized agent
Damage caused by accidental misuse
Damage caused by disaster
Common replacement parts including platinum wire
Damage caused by use of organic solvents or basic detergents
For inquiries or to request repair service, contact your local Bio-Rad office.
Warranty information:
Model:
Catalog number:
Date of delivery:
Lot number:
Invoice number:
Purchase order number:
13
4006216 Rev A
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