Stripping and Re-probing membrane
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 dH2O
o Warm up to 65°C in microwave, then add 600 ml of b-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.
MILD STRIPPING (adapted from AbCam protocol)
Solutions
o Mild stripping buffer:
Ø 15 g glycine
Ø 1 g SDS
Ø 10 ml Tween 20
Ø Adjust pH to 2.2 with HCl
Ø 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