1. acrylamide: bis-acrylamide （29：1）, 购买于“生工， 货号： B546017-0500
2. TEMED;
3. APS(过硫酸铵)；
4. Tris base;
5. SDS;
6. Glycerol;
7. Coomassie Blue Stain R-250
8. Methanol (甲醇)
9. Acetic acid（醋酸）;
Part I
1. Resolving Gel
The following recipe is based on 30%(w/v) acrylamide stock solution (enough for 3 small gels)
6% | 8% | 10% | |
solution A (ml) | 3 | 4 | 5 |
solution B (ml) | 3.75 | 3.75 | 3.75 |
H2O (ml) | 8.25 | 7.25 | 6.25 |
TEMED (μl) | 10 | 5 | 5 |
10% APS (μl)** | 75 | 75 | 75 |
Total volume (ml) | 15.085 | 15.08 | 15.08 |
to make 10% small SDS-PAGE gel
1 gel | 2 gels | 3 gels | |
solution A (ml) | 1.67 | 3.34 | 5 |
solution B (ml) | 1.25 | 2.5 | 3.75 |
H2O (ml) | 2.083 | 4.166 | 6.25 |
TEMED (μl) | 1.67 | 3.34 | 5 |
10% APS (μl)** | 25 | 50 | 75 |
Total volume (ml) | 5.03 | 10.06 | 15.08 |
** Add the APS last. Once added, it will start to polymerize.
2. 4% Stacking Gel
1 gel | 2 gels | 3 gels | |
solution A (ml) | 0.2 | 0.4 | 0.6 |
solution B (ml) | 0.5 | 1 | 1.5 |
H2O (ml) | 1.32 | 2.64 | 3.96 |
10% APS (μl) ** | 13.33333 | 26.66667 | 40 |
TEMED (μl) | 3.333333 | 6.666667 | 10 |
Total volume (ml) | 2.036667 | 4.073333 | 6.11 |
** Add the APS last. Once added, it will start to polymerize.
3. Working Solution
Solution A: acrylamide: bis-acrylamide （29：1）, 购买于“生工， 货号： B546017-0500
”keep in 4 degree
Solution B: 4x Separating gel buffer
dissolve 91 g Tris base in 300ml H2O |
adjust pH to 8.8 with about 13.5ml concentrated HCl |
add 2 g SDS |
reach 500ml with H2O and filter |
Solution C：4xStacking Buffer
dissolve 6.05 g Tris base in 40ml H2O |
adjust pH to 6.8 with about 4.1ml concentrated HCl |
add 0.4 g SDS |
reach 500ml with H2O and filter |
10% APS (keep in -20 degree)
0.5g Ammonium Persulfate |
5ml H2O |
6xSDS protein loading buffer, 20ml (keep in -20 degree)
12ml | glycerol |
2.4g | SDS |
6 ml | Tris-Hcl pH 6.8 |
0.0012g | Bromophenol blue |
4. 1x Protein Running Buffer, 1 liter
Tris base | 3 g |
Glycine | 14.5 g |
20%SDS | 5 ml |
Part II: making SDS-PAGE gel and gel running
1. making small SDS-PAGE gels
A). for each SDS-PAGE gel, sandwich one small and one large glass plate, separated by spacers and an alignment card
B). Slide into holder without tightening the screws
C). Place on pouring stand, making sure that the glass plates are pushed all the way to the bottom before tightening the screws. If the assembly is correct, then the whole set-up should snap into place when placed above the gray gasket
D). Remove alignment card
E). pour resolving gel
F). add 1-2ml water to the top of the resolving gel
G). let polymerize for 10-15 min
H). Invert gel to remove all water on the top
I）Pour stacking gel to fill the remaining space between the glass plates
J) insert comb and let polymerize for 10-15min
K) mix protein loading dye with protein samples, boil samples 5-10min
L) place gel in holder/electrode, then transfer to running tank, add protein running buffer
M) use protein running buffer to clean the walls
N) loading protein samples
2. Protein Running
Run at 150V through the stacking gel, and then run 110Vx1~1.5hour for small gels until the dye front reaches the bottom of the gel
Part III: Coomassie Blue (Brilliant Blue) Staining (Optional)
***Do not do Part III, if you are planning to do Western Blot
1. Stain the protein gel with Coomassie Blue Buffer for at least 1 hour at 50 degree in a covered box. The longer the incubation, the better
Coomassie Blue Stain Buffer | |
Coomassie Blue Stain R-250 | 1.25 g |
Methanol | 0.5L |
Acetic Acid | 0.1L |
ddH2O | 0.4L |
** Coomassie Blue Stain Buffer can be recycled and used for many times.
2. Wash with Destaining Solution I (50% Methanol, 7% HAc), twice, 30 minutes for each time, at 50 degree
3. Wash with Destaining Solution II (5% Methanol, 7% HAc), a few times, with the final wash being done overnight. All washes are done at RT.
Part IV: Western Blot
1. Materials:
1x TGM (Transfer Buffer), chill buffer at 4 ̊C, and keep at 4 ̊C
1xTGM | |
glycine (Bio-Rad) | 7.206 g |
Tris Base | 1.514 g |
Methanol | 100ml |
add dd H2O to reach 500ml |
1xTBST
NaCl | 7.3 g |
Tris base | 3.028 g |
Tween 20 | 1 ml |
pH 8.0 | add about 1.4 ml concentrated HCl |
add ddH2O to 1 liter |
2. Cut off stacking gel and nick top left-hand corner of resolving gel for orientation
3. Measure the dimensions of the gel and note the positions of the ladder bands
4. Transfer gel, while still attached to glass plate, to box containing TGM and peel off gently with a spatula
5. Agitate 15-20 minutes at RT to remove salts and SDS
6. Cut a piece of nitrocellulose membrane to the size of the gel and mark and/or clip one corner as the top left-nad corner. Handleonly with flat forceps
7. Immerse membrane in TGM for 10-15 minutes
8. Cut 2 pieces of >=3 mm filter paper to the dimensions of the gel
9. Open a gel holder cassette in a casserole dish, black side down and hinges to the left and below the black side
10. Soak a fiber pad with TGM and place in the center of the black side
11. Soak one piece of filter paper with TGM and place on top of the fiber pad
12. Roll out bubbles with glass tube and add 3 ml of TGM onto the top
13. Pour out old TGM and add fresh TGM to gel
14. Fish gel out with a glass plate
15. place gel on top of filter paper
16. Roll out bubbles with glass tube and add 3 ml of TGM onto the top
17. Place membrane on top of gel, with the gel's top left mark facing the membrane's top left mark.
18. Rll oout bubbles with glass tube and add 3 ml of TGM onto the top
19. Soak a second piece of filter paper with TGM and place on top of the membrane
20. Roll out bubbles with glass tube and add 3 ml of TGM onto the top
21. Soak a second piece of fiber pad and place on top of stack
22. Roll out bubbles with glass tube and add 3 ml of TGM onto the top
23. Close the gel holder cassette.
24. Place in a transfer tank (orient the white and black sides of the cassette with the white and black panels of the electrode) and fill with TGM
25. Place the tank in a styofoam box containing ice
26. Run 30 minutes at 25V
Part V: Immunodetection
1. (Optional) Rinse blot with ddH2O several times to remove Methanol and salts, and stain all protein bands with 0.5% Ponceau S buffer. Take a picture and rinse off the Ponceau S with 1xTBST.
0.5% Ponceau S buffer, 100 ml | |
Ponceau S | 0.5 g |
Glacial Hac | 1 ml |
ddH2O | 99 ml |
** 0.5% Ponceau S buffer can be recycled and used for many times.
2. Following Western Blot transfer, place membrane in a box containing 10 ml 5% nonfat milk in TBST for 1 hour at RT.
3. Wash briefly with 1xTBST
4. Wash again with 1xTBST, twice, 5 minutes/time
5. Place blots in box containing 8ml primary antibody (use 1: 500 to 1:5000 dilutions) in 1xTBST and incubate at 4 degree overnight.
6. Wash blots with 1xTBST, twice, 15 minutes/time
7. Replace blot in box containing 10 ml second antibody (Use 1: 1000 to 1:5000 dilutions) in 1xTBST and incubate for 30 minutes
8. Wash blots with 1xTBST, twice, 15 minutes/time
9. Rinse twice with ddH2O for 1 minute to remove the Tween-20
10. Place membrane on Saranwrap, dry it
11. Add ECL-Plus detection solution
12. Incubate for 5 minutes at RT. This is best done in the dark. Ensure that the entire membrane is covered by detection solution, detect signals by X-film or Chemiluminescence Detector.
