HARSH STRIPPING (from Boston Bioproducts)
Solutions: o Make 250 ml TBS/T o 4x Membrane Stripping Buffer (Boston Bioproducts, Cat # BP-96) dilute to 1X: for 100 ml
25 ml 4x stripping buffer
75 ml dH
2
O o
Warm up to 65

C in microwave, then add 600
 l of

-mercaptoethanol in hood; mix well.
Procedure:
1.
Wash membrane in 50 ml hot stripping buffer for 5 minutes (gently rock the container in hood)
2.
Submerge the membrane in 50 ml hot stripping buffer and incubate at 65

C for 30 minutes with occasional agitation (use heated oven).
3.
Wash the membrane for 5x5 min in TBS-T at RT, using large volumes of wash buffer.
4.
Detect using detection protocol (for at least 30 minutes).
5.
Wash the membrane with 1X TBS for 5 minutes
6.
Block the membrane in 5% non-fat dry milk in TBS-T for 1 hr at RT
7.
Incubate with new primary antibody; following day, secondary antibody, and detection.
Updated 04/08/10 by EA

MILD STRIPPING (adapted from AbCam protocol)
Solutions o
Mild stripping buffer:

15 g glycine

1 g SDS

10 ml Tween 20

Adjust pH to 2.2 with HCl

Bring volume up to 1 L with ultrapure water. o
Make 200 ml TBS/T o
Make 200 ml TBS
Procedure
1.
Use a volume of mild stripping buffer that will cover the membrane (50 mL for a big membrane). Incubate at 37

C for 15 minutes.
2.
Discard buffer.
3.
Repeat step 1 and 2 for 4 times
4.
Wash twice 10 minutes in abundant TBS (100 mL)
5.
Wash twice 5 minutes in abundant TBST (100 mL)
6.
Detect using detection protocol (for at least 30 minutes).
7.
Wash the membrane with 1X TBS for 5 minutes
8.
Block the membrane in 5% non-fat dry milk in TBS-T for 1 hr at RT
9.
Incubate with new primary antibody; following day, secondary antibody, and detection.
Updated 04/08/10 by EA