IPBuffer

IP Buffer

To PBS add,

10mM EDTA

.1%Triton-X 100

1mM PMSF

(To make 50mls, add 1ml of .5mM stock EDTA, 0.5ml of 10% stock of Triton-X,

and .5ml of 100mM stock of PMSF) Thaw PMSF before using

1X-TBST

20mM Tris

150mM NaCl

.1% Tween 20, P.H 7.4

Blocking Buffer

8. Remove the sup from the beads *****this is what you want to run in the gel

V. Run Gel

1. Remove comb from the gel and wash out wells

2. Dry and mark the bottom of the wells

3. Remove the paper tab

4. Inrt with the gels with the ridge facing the center of the apparatus, and lock

down

5. Fill inner chamber with SDS running buffer

6. Fill ½ of the outer chamber with SDS running buffer

7. Put 20ul of marker and protein in the wells

8. Turn the current all the way up, and t the voltage to 100 v—after it gets

through the stacking gel, t it to 150 v

VI. Transfer

1. Crack open the gel apparatus

2. Let gel stick to one side

3. Remove the stacker gel

4. Put gel in transfer buffer by using gravity

5. Let gel equilibrate in buffer for 15-20 minutes

6. While the gel is equilibrating, put transfer paper in methanol to wet

7. Pick transfer paper up with hands and put in transfer buffer

4. Put the transparency in the film developer and put the transfer paper in the

transparency

5. Develop film using a film developer.