Western Blot Probing by ECL

1. After electroblotting of protein gel to immobilon membrane (See Novex NuPAGE method), mark the side of the membrane not in contact with the gel with permanent ink and cut a small corner piece off the bottom of the membrane. This will aid in determining which side of the membrane has protein bound and the orientation of the filter.

2. Use forceps to transfer the membrane to a generous amount of TBST + 7.5% milk (TBSTM). Incubate with gentle shaking for 60 min. at room temp. or overnight at 4 degrees (after shaking a few minutes at room temp, the membrane can be left in the fridge overnight without shaking). This step blocks the membrane to prevent non specific sticking of the antibodies.

3. Rinse the membrane with TBSTM and place the membrane with protein side down in a minimal volume of TBSTM (3-5 ml in a square petri dish works well).

4. Add antisera or antibody at the desired dilution (typically 100-5000 fold dilution dependent on the specific antibody). Incubate at room temperature with vigorous shaking for the desired time (typically 1-2 hrs)

5. Dump off the TBSTM + antisera and rinse the membrane with TBSTM three times taking care to remove as much solution as possible after each rinse.

6. Wash the membrane three times with a generous amount of TBSTM. During each wash incubate 10 min at room temp with gentle shaking.

7. After the three washes, resuspend the membrane in a minimal volume of TBSTM (3-5 ml). Add the appropriate HRP conjugated secondary antibody at the desired dilution (typically 2000-fold dilution). Incubate 60 min with vigorous shaking.

8. Dump off the TBSTM + antibody and rinse the membrane with TBST (no milk) three times taking care to remove as much solution as possible after each rinse.

9. Wash the membrane 3-4 times with a generous amount of TBST. During each wash incubate 10 min at room temp with gentle shaking.

10. Make up the working solution of Pierce Supersignal ECL reagent by mixing equal volumes of the two solutions (typically 1.5 ml of each solution for a small western blot).

11. Remove the membrane and briefly blot on filter paper, but do not dry the membrane. Turn membrane side up on a piece of saran wrap. Pipet the ECL working solution on the protein side of the membrane. Incubate 3-5 minutes.

12. Carefully drain excess ECL solution by blotting up the excess solution from the side of the blot and seal the blot in saran wrap. Expose to film for the desired time (typically 30 sec to 10 min depending on the antibody).

13. If the membrane will be reprobed in the next day or two without stripping, wash several times in TBST and store at 4 degrees in TBST.

10X TBS		1 liter
200 mM Tris pH7.6		24.2 g Tris
1.37 M NaCl		80 g NaCl

pH to 7.6 with HCl and store at room temp.

TBSTM		300 ml
10 X TBS		30 ml
non fat dry milk		22.5 g
Tween 20		0.3 ml
		250 ml H2O

Adjust pH with to 7.6 with 8M NaOH

TBST		300 ml
10 X TBS		30 ml
Tween 20		0.3 ml
		270 ml H2O

Stripping the antibody from Western Blot

(Natalya Yudkovsky and Jeff Ranish)

Note: these two methods both work, but the first is less time consuming:

Easy Stripping Method:

1. Put the blot in the following solution:

100 mM 2-mercaptoethanol

2% SDS

62.5 mM Tris-Cl pH 6.7

2. incubate 50 degrees for 30 min. in a sealed plastic container in the shaking water bath. (this can be repeated if necessary)

3. Rinse with TBST. Can be stored at 4 degrees.

4. Reblock with TBSTM as in step 2 of Western probing method (above) before reprobing with antibody.

Harder Stripping Method:

1. Put the blot in the following solution:

0.2 M Glycine-HCl pH2.5

0.05% Tween-20

100 mM 2-mercaptoethanol

2. incubate 60 degrees for 60 min. in a sealed plastic container in the shaking water bath.

3. Repeat.

4. Rinse with TBST. Can be stored at 4 degrees.

5. Reblock with TBSTM as in step 2 of Western probing method (above) before reprobing with antibody.