SDS -PAGE Gel and Western Blot

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)

to make 10% small SDS-PAGE gel

** Add the APS last. Once added, it will start to polymerize.

2.   4% Stacking Gel

** 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

Solution C：4xStacking Buffer

10% APS (keep in -20 degree)

6xSDS protein loading buffer, 20ml   (keep in -20 degree)

4.  1x Protein Running Buffer, 1 liter

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  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

   1xTBST

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 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.