Bio-Rad Comparative Proteomics Kit II Western Blot Module User Manual

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