25
Quick Guide
1ml
Comparative Proteomics Kit II:
Western Blot Module – Quick
Guide
Lesson 1 Quick Guide
1 Label one 1.5 ml fliptop micro tube for each of
five fish samples. Also label one screw-cap micro
tube for each fish sample.
2. Add 250 µl of Bio-Rad Laemmli sample buffer to
each labeled fliptop microtube.
3. Cut a piece of each fish muscle about 0.25 x 0.25
x 0.25 cm
3
( ) and transfer each piece into a
labeled fliptop micro test tube. Close the lids.
4. Flick the microtubes 15 times to agitate the tissue
in the sample buffer.
5. Incubate for 5 minutes at room temperature.
6. Carefully transfer the buffer by pouring from each
fliptop microtube into a labeled screw-cap
microtube. Do not transfer the fish!
7. Heat the fish samples in screw-cap microtubes
for 5 minutes at 95°C.
Lesson 2 Quick Guide
1. Set up Mini-PROTEAN 3 gel box.
2. Prepare a Ready Gel cassette by cutting
along the black line on the bottom of the
cassette with a razor blade and pulling off
the plastic strip, as indicated on gel
cassette.
3. Remove the comb from the Ready Gel
cassette.
4. Place Ready Gel cassette into the
electrode assembly with the short plate
facing inward. Place a buffer dam or
another Ready Gel cassette on the
opposite side of the electrode assembly,
with notch on buffer dam facing inward.
QUICK GUIDE
5. Slide gel cassette, buffer dam, and
electrode assembly into the clamping
frame.
6. Press down the electrode assembly while
closing the two cam levers of the
clamping frame.
7. Lower the inner chamber into the mini
tank.
8. Completely fill the inner chamber with 1x
TGS gel running buffer, making sure
the buffer covers the short plate (~150 ml).
9. Fill mini tank approximately 200 ml of
1x TGS gel running buffer.
10. Plate sample loading guide on top of the
electrode assembly.
26
Quick Guide
QUICK GUIDE
Pipet Tip
Sample
Loading
Guide
27
Quick Guide
11. Heat fish samples and actin and myosin
standard to 95°C for 2–5 min.
12. Load your gel:
Lane Volume Sample
1 & 2 empty Empty
3 5 µl Precision Plus Protein
Kaleidoscope
prestained standards
4 5 µl fish sample 1
5 5 µl fish sample 2
6 5 µl fish sample 3
7 5 µl fish sample 4
8 5 µl fish sample 5
9 5 µl actin and mysin standard
(AM)
10 empty empty
13. Electrophorese for 30 minutes at 200 V in 1x
TGS gel running buffer.
14. Proceed directly to Lesson 3, continue to step 15
of Lesson 2 to stain gels or store gels overnight
at 4°C.
15. If the gels are to be stained, save 50 ml of 1x TGS
gel running buffer.
16. Remove gel from cassette and transfer gel to a
container with 25 ml Bio-Safe Coomassie
stain/per gel and stain gel for 1 hour, with gentle
shaking for best results.
17. Discard stain and destain gels in a large
volume of water for at least 30 minutes to
overnight, changing the water at least once.
Blue-stained bands will be visible on a clear gel
after destaining.
QUICK GUIDE
28
QUICK GUIDE
Quick Guide
Banana Plugs
Anode Banana Plug
(Red)
Notch on
U-Shaped Gasket
Gel Sandwich
Pressure Plate
Cams
Mini Tank
Inner
Chamber
Assembly
Clamping
Frame
Electrode
Assembly
Cathode Banana Plug
(Black)
Lid
Fig. 4. Assembling the Mini-PROTEAN 3 cell.
Lesson 3 Quick Guide
1. Using a ruler, chop the top and bottom off
the gel.
2. Equilibrate the gel in blotting buffer for
15 minutes on a rocking platform.
3. Soak fiber pads thoroughly in blotting buffer.
4. Mark the white nitrocellulose membrane
with penciled (or black ball point pen) initials
and prewet in blotting buffer along with the
blotting paper.
5. Make the blotting sandwich:
a. Add 1 cm depth of blotting buffer to
container and insert plastic cassette
with black side down.
b. Lay a wet fiber pad on the black side of
the cassette.
c. Lay one wet blotting paper on the fiber
pad and roll out air bubbles.
d. Lay gel squarely on blotting paper and
roll out air bubbles.
e. Lay wet nitrocellulose membrane on the
gel and roll out air bubbles.
f. Lay one wet blotting paper on the
membrane and roll out air bubbles.
g. Lay a wet fiber pad on top of the blotting
paper.
h. Close the cassette and clamp together
with the white clip.
29
Quick Guide
Fiber pad
Blotting paper
Membrane
Gel
Blotting paper
Fiber pad
QUICK GUIDE
Quick Guide
6. Set up the Mini Trans-Blot module with the
black side of the cassette next to the black
side of the Mini Trans-Blot module. Add a
frozen Bio-Ice module and fill with blotting
buffer up to the white clip.
7. Place lid on tank, matching the power
cords red-to-red and black-to-black, then
blot at 20 V for 2.5 hours.
8. At this point the blots can be stored in the
tanks submerged in blotting buffer at room
temperature overnight or the sandwiches
dismantled and the blots placed in blocker
overnight at 4°C.
30
QUICK GUIDE
Fig. 5. Assembly of the Mini Trans-Blot cell.
31
Quick Guide
Electrode
module
Lid
Fiber pad
Blotting paper
Membrane
Gel
Blotting paper
Fiber pad
Gel holder
cassette
Bio-Ice
cooling
unit (keep
frozen at –20°C)
Buffer tank
QUICK GUIDE
Lesson 4 Quick Guide
1. If not blocked overnight, immerse
membrane in 25 ml blocking solution for
15 minutes to 2 hours at room temperature on a rocking platform.
2. Discard blocking solution and incubate
membrane with 10 ml of primary antibody
for 10–20 minutes on rocking platform set
to a faster setting to ensure constant
coverage of the membrane.
3. Quickly rinse the membrane in 50 ml of
wash buffer then discard the wash.
4. Add 50 ml of wash buffer to membrane for
3 minutes on rocking platform at a
medium speed setting.
5. Discard the wash and incubate membrane
with 10 ml of secondary antibody for
5–15 minutes on rocking platform set to
a fast setting.
6. Quickly rinse the membrane in 50 ml of
wash buffer and discard the wash.
7. Add 50 ml of wash buffer and wash
membrane for 3 minutes on rocking
platform on a medium speed setting.
8. Discard the wash and add 10 ml of HRP
color detection reagent.
9. Incubate 10–30 minutes, either with
manual shaking or on a rocking platform,
and watch the color development.
10. Rinse the membrane twice with distilled
water and blot dry with paper towel.
11. Air dry for 30 minutes to 1 hour and then
cover in plastic wrap or tape in lab book.
32
Quick Guide
QUICK GUIDE
Bio-Rad
Laboratories, Inc.
Life Science
Group
Bulletin 5478 US/EG Rev A
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