Hoefer PR625 User Manual
user manual
ImmunoBlot and
ImmunoBlot XL
Operating instructions
um PR645-IM/Rev.B0/08-12
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Introduction ..........................................................1
Unpacking and inventory ....................................2
Instructions ...........................................................3
Select and block membrane ............................... 3
Load membrane ................................................3
Aspirate and introduce antibody solutions ............ 4
Incubate ........................................................... 4
Remove primary antibody solutions
and wash the blot ..............................................5
Introduce secondary antibody and incubate .........6
Remove and develop the blot..............................7
Clean the ImmunoBlot .......................................7
Troubleshooting ..................................................... 8
Appendix A: Technical notes ................................. 10
Appendix B: Detection using ECL Western
blotting detection systems ....................................16
Ordering information ............................................18
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Introduction
Transfer of proteins or nucleic acids, fractionated by gel electrophoresis, to an immobilizing
membrane surface increases the sensitivity of
a wide range of detection methods, reduces
time of analysis and makes sequential probing
possible. In particular it has made possible use
of immunological procedures that are not practical to carry out in a gel. To assay material on a
sheet of membrane with different probes simultaneously commonly requires first cutting the
membrane into a number of parallel strips. This
procedure however destroys the correspondence
between different lanes or positions on a gel.
The ImmunoBlot® and ImmunoBlot XL
preserve this correspondence while minimizing the volume of detection reagents needed for
individual lanes. Two clear plastic plates, one
smooth and one with channels, clamp a 15 ×
15 cm transfer membrane to define and separate
the original gel lanes into individual channels.
These separate channels allow for the use of
different detection reagents (primary antibodies,
conjugated secondary antibodies, antigens and/
or reaction substrates) in each channel.
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There are 2 principal applications for the
ImmunoBlot and ImmunoBlot XL:
Multiple antisera or probes may be tested
against a single sample of protein or nucleic
acids. The single sample is loaded across the
entire top of a slab gel, then run and blotted to
a membrane. When clamped to the ImmunoBlot,
the membrane can be tested against multiple
antisera or probes.
A single antiserum or probe can be tested
against multiple antigens or nucleic acids.
The two models differ in the number and
volume of the reaction channels. The ImmunoBlot has 25 channels that hold a maximum
volume of 250 μl each. The ImmunoBlot XL has
45 channels that hold a maximum volume of
140 μl each.
Unpacking and inventory
Unwrap all packages carefully and compare
contents with the packing list, making sure all
items arrived. If any part is missing, contact
your local sales office. Inspect all components
for damage that may have occurred while the
unit was in transit. If any part appears damaged,
contact the carrier immediately. Be sure to keep
all packing material for damage claims or to use
should it become necessary to return the unit.
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Instructions
The following detail the steps for probing a
membrane using the ImmunoBlot.
Select and block membrane
1
Select a membrane of choice (nitrocellulose,
Hybond ECL™; PVDF, Hybond P; low fluorescent PVDF,
Hybond LFP) for the assay and cut to:
Length: 15 cm
Width: Cut to accommodate channels to be used
Maximum width for all channels: 15 cm
2
Block membranes with excess non-specific protein.1
3
Note: If blocked membranes have been stored in a
protein-free solution, re-block them for 1 to 2 minutes
in a tray of blocking solution.
Load membrane
1
Remove the top acrylic plate of the ImmunoBlot
and turn it over.
1
See Appendix A, Note 1,
Blocking the membrane.
2
See Appendix A, Note 2,
Aligning the membrane.
2
With the antigen-bearing face of the membrane facing
the ImmunoBlot channels, position the membrane so
it covers all the channels.
2
3
Place a new, dry sealing pad (supplied with unit) over
the membrane.
4
Set the bottom plate of the unit on the inverted top
plate so the alignment pins fall in place.
5
Ensure there is no gap between the top and
bottom plates.
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Aspirate and introduce antibody solutions
Recommendation: Use
a disposable pipette tip
connected to a water
vacuum aspirator for this
purpose.
Important! Take care not
to touch the pipette tip
anywhere on the unit except
in the desired sample hole.
1
Aspirate excess liquid from the channels through the
numbered holes.
Note: To avoid drying of the membrane, channels
should be loaded within five (5) minutes of aspirating.
2
Pipette antibody solution through the numbered holes.
Press the pipette tip firmly into each hole and inject
the liquid rapidly in a single smooth action until the
channel is filled. Take care not to introduce bubbles.3
3
Add buffer to all unused channels that cover the
membrane.
number of approximate
model channels channel volume4
ImmunoBlot 25 250 µl
ImmunoBlot XL 45 140 µl
Incubate
1
Place the unit on a rocking platform with the channels
aligned in the direction of rocking.
Note: For best results, use a slow rocking speed (5 to
6 tilt cycles per minute). A gyratory shaking platform
is not effective for this purpose.
3
See Appendix A, Note 3,
Eliminating bubbles.
4
See Appendix A, Note 4,
Optimal volumes.
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2
Incubate the unit on the rocking platform for 30 to
60 minutes at room temperature.
Remove primary antibody solutions and wash the blot
Use the washing manifold to remove the
antibody solution and wash the blot.
1
Position the 2 identical parts of the washing manifold
in the slots on either side of the top plate and press
firmly until the O-rings are seated in the slots.5
2
To aspirate all samples simultaneously, first connect
pieces of the tubing supplied with the unit to both
parts of the manifold using a luer fitting. Now,
connect the open end of the tubing from the manifold
piece to a trap connected to a vacuum source.
Then place the open end of the tubing from the
second manifold piece into a beaker containing
wash buffer.
When you start the vacuum antibody solutions in the
channels are removed and the wash buffer is drawn
in from the other side. Please refer to your Western
detection protocol for wash instructions. For Western
detection we recommend at least 2 quick washes
in buffer followed by two longer washes in buffer
(5 min while rocking is sufficient) before introducing
the secondary antibodies.
5
See Appendix A, Note 5, Fitting
the O-rings into the slots.
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