Western Blotting
Western Protocol
9/30/2009 Maggie
Day1
Gel running
1. Prepare the running buffer.
(For 1L: 900ml ddH2O + 100ml of 10X Tris/Glycine/SDS buffer)
2. Set up the gel. (10% premade PAGE gel-BioRad, stored at 4°C),
3. Pour the running buffer into the setup (make sure there is no leak), about 80%.
Fulfill the middle of the setup with running buffer.
Don’t fill the outside chamber in order to see the loading wells clearly.
4 Take out the comb carefully.
5. Load 10 ul of the following samples.
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1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
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positive control |
ladder |
sample1 |
sample2 |
sample3 |
sample4 |
sample5 |
sample6 |
Sample7 |
Sample8 |
6. Fulfill the whole set up with running buffer.
7. Connect the set up to power supply and run at 95V for 1-1.5 hr.
Usually, stop running when the blue line is at the bottom of the gel.
Transfer
8. Prepare the transfer buffer. (Old method)
(50ml 10X Tris/Glycine buffer + 750ml ddH2O + 200ml methanol)
9. Incubate 2 sponges, a set of paper and membrane and 2 sponges into the transfer buffer in order. (Tips: Use the knife to immerse them. The transfer buffer is stored at 4°C).
Blocking
10. Prepare the Blocking solution 5% milk in 1X TBST.
(1 X TBST + 5g non-fat milk) 5% milk and stir for 1 hr.
11. Pour off the buffer, take out the entire sandwich.
Take out the Gel sandwich with the two glasses.
Use knife to separate the thicker glass (well-side up in the palm) carefully with the gel and thin glass. Keep the knife’s sharp side out of the arm.
Use the top side of the knife to cut off the row on top and bottom of gel.
12 Transfer the gel onto the membrane using the following sandwich procedure.
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1 |
negative plate (container) |
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2 |
2 pieces of sponge |
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paper |
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gel |
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membrane |
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paper |
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2 pieces of sponge |
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cover with positive plate (plain) |
(Add buffer between the sandwiches, use the knife to push the upper side of the sponge in order to make sure there are no bubbles in-between.)
12. Add transfer buffer into the sandwich space.
13. Put ddH2O into the outside space to fulfill the setup.
14. Connect the set up to 30V power supply and run it for 1 hr.
15. Take out the membrane and incubate with blocking solution for 1 hour in shaker (66rpm).
19. Prepare the 1°Ab (usually 1/1000:1ml of blocking solution + 1ul of 1° Ab)
20. Put the membrane on a piece of paper. Then put it into a plastic bag, add the 1°Ab into the bottom of the bag, seal the bag. Make sure no bubbles in the bag.
21. Incubate with 1°Ab on the shaker at 4°C cold room overnight.
Day2
1. Prepare TBST buffer (0.05% Tween in 1X TBS) ( i.e. washing buffer)
(1800 ml H2O+200 ml 10X TBS+10 ml Tween 20)
(1000ml 1X TBS + 5ml Tween 20)
2. Wash: each time change the 1X TBST.
Quick wash the membrane with 1X TBST for 5-7 times.
Wash with 1X TBST for 10 min with shake, twice.
Wash with 1X TBST for 5 min with shake, twice.
Quick wash with 1X TBST for 5-7 times.
3. Prepare 2° Ab: 20ml /membrane.
20 ml 1X TBST +2 ul 2°Ab of HRP-Goat anti Rabbit or Mouse.
Incubate with 2 Ab for 30 min.
4. Repeat step2.
5. Put membrane into the black box.
a. Put membrane on a piece of paper, then on a plastic wrap.
b. Put 500ul detecting reagent A and 500ul reagent B into Eppendorf tube, (Remember to change the tip.) mix well.
c. Add 1 ml detecting complex on top of the membrane, wait for 1 min.
d. Put membrane on a piece of paper to dry the unwanted solution.
e. Cover the membrane with plastic wrap in the black box.
f. Cover it and get out of the bubbles.
6. Develop the film.
Carry the black box, films and timer, go to 10th floor darkroom.
Tips: Fold the left upper corner of the film to distinguish the direction of the film and the membrane.
(Exposure time: 5 sec, 10 sec, 30 sec, 1 min, if no signal, 20 min. If no signal at 20 min, no signal at all.)
7. Strip the membrane and reblot.
7.1. Prepare the Blocking solution 5% milk in 1X TBS.
7.2 Quick Wash the membrane with 1X TBST for 5 times.
7.3 Add 20 ml of 1 X reblot buffer (18 ml+ 2 ml 10 X reblot buffer). 10 X reblot buffer is stored at 4°C.
7.4 Shake 15 min.
7.5 Quick Wash the membrane with 1X TBST for 5 times.
7.6 Incubate 10 min with shake.
7.7 Wash the membrane with 1X TBST for 5 times.
7.8 Block with 5% milk.
7.9 Prepare the 1°Ab (usually 1/1000:1ml of blocking solution + 1ul of 1° Ab)
7.10 Put the membrane on a piece of paper. Then put it into a plastic bag, add the 1°Ab into the bottom of the bag, seal the bag. Make sure no bubbles in the bag.
7.11 Incubate with 1°Ab on the shaker at 4°C cold room overnight.
2009 cancer research Role of Cyclin D1 as a Mediator of c-Met– and B-Catenin–Induced Hepatocarcinogenesis
Preparation of lysates and Western blotting. Liver tumors were frozen on dry ice on harvesting, and homogenates were sonicated in lysis buffer [150 mmol/L NaCl, 1.0% Igepal, 0.5% sodium deoxycholate, 0.1% SDS,50 mmol/L Tris (pH 8.0)] and centrifuged at 14,000 rpm at 4jC. Supernatant was boiled in Laemmli sample buffer for Western blot analysis. The antibodies used are as follows: anti-CCND1 (Ab3, 1:1,000, Lab Vision;C-20,1:500, Santa Cruz Biotechnology), anti-CCND2 (M-20, 1:500, Santa CruzBio technology), anti-CCND3 (C-16, 1:300, Santa Cruz Biotechnology), anti-Cdk4 (C-22, 1:500, Santa Cruz Biotechnology), anti-Cdk6 (1:300, Santa Cruz Biotechnology), anti-Cdk2 (M-2, 1:500, Santa Cruz Biotechnology), anti-GS(1:1,000), anti-actin (1:5,000, Sigma), anti-ERK (1:1,000), anti-phospho-ERK(1:1,000), anti-phospho-Met (1:1,000, Cell Signaling), and anti-V5 (1:5,000, Invitrogen). Western blots were quantified using the ImageJ software.7