Western Blotting

1) Run protein gel.

Set up blot:

2) Prepare two tupperware containers, one with dH2O and one with 1/2X Transfer Buffer (TB).

3) Remove one glass plate from the gel by twisting a spacer between the plates. To remove the other plate, place the gel in the TB container and gently scrap off with a flat scoopula. Let the gel soak for about 5 mins.

4) To make the blotting sandwich, begin by putting the blotting apparatus in the TB container with the black side down. Then put on a pad and one piece of paper (make sure both are completely wet in the TB). Next the gel goes on.

5) Use the tweezers to grab the nitrocellulose membrane and dunk it in the dH2O container, make sure the entire membrane is wet, and then move it to the TB. When it is completely soaked with TB, move it onto the sandwich next.

6) To complete the sandwich, one more piece of paper goes on and then the last pad, again making sure both are thoroughly soaked in TB.

7) Roll a 15ml tube up and down and side to side over the completed sandwich to remove any bubbles. Close the blotting apparatus securely.

8) Put a small stir bar in the gel box underneath the black and red sleeve. Then put the ice pack in next to the sleeve.

9) Put the blotting sandwich into the black and red sleeve with the black part of the sandwich facing the black part of the sleeve. Then fill the gel box up to the top of the red half of the sleeve with TB.

10) Run the blot on manual and constant V at 100V for about 1 hour.

Staining the blot:

11) Dump out the TB, and remove the sandwich.

12) Use tweezers to put membrane into a small tupperware top and cover with ponso (red stuff). Leave on the orbital shaker or rocker for about 15 mins.

13) Rinse with water until markings appear. Use a pencil to mark the ladder, and distinguish the 50kDa marker somehow. Cut the top right-handcorner so that "up" is easily recognizable.

14) Wash with block 10-15 mins. on the rocker.

15) Transfer to the smallest container possible.

Binding the antibody

16) Add diluted primary antibody.

17) Incubate primary antibody overnight at 4 C.

18) Wash 3 times for 15 mins. each with 1XTBST on a rocker.

19) Dilute out secondary antibody 1: 5,000 (1┬Ál in 5ml).

20) Dump off third wash and put in secondary antibody mix. Incubate 1 hr. on a rocker.

21) Wash 3 times for 15mins. each with 1XTBST on a rocker.

Developing the blot:

22) Materials needed: Two pieces of saran-wrap, detection reagent 1 and 2 (bottles at 4 C with a big ECL on the label, one with a white top and one with a black top), film cassette, scissors, tweezers and film.

23) Mix 2ml of 1 and 2ml of 2 in a 15ml tube (BE SURE TO CHANGE PIPETS IN BETWEEN REAGENTS).

24) Use tweezers to move membrane on to a piece of saranwrap and drip the reagent mix all over the membrane. Let stand for 1min.

25) Drip off excess mix, and transfer the membrane to a clean piece of saranwrap upside down. Wrap the membrane up in the saranwrap and place it into the film cassette right-side up.

26) Bring the cassette, film and scissors in a darkroom with a developer. When in the darkroom, cut a piece of film to fit the cassette.

27) Burn the film for 1min, bend the top right hand corner down and back again to distinguish the correct orientation.

28) Slide the film through the developer to develop.