Griffin:Membrane Stripping and Reprobing
Following detection of proteins with enhanced chemiluminescence (ECL), membranes can be stripped of bound antibody and reprobed with a different antibody. If you plan on stripping and reprobing the membrane, the membrane should be stored moist and wrapped in plastic wrap at 2-8°C after each immunodetection.
The key to a successful stripping and reprobing is to rinse the membrane until there is no beta-mercaptoethanol odor present. This will require several initial shake rinses into a proper waste storage container, followed by rinsing under milli-q water for a few minutes at a sink, then a few more rinses in TBS or PBS. Check for the odor and when it is gone, the membrane is ready.
- 62.5 mM Tris-HCl, pH 6.7,
- 100 mM beta-mercaptoethanol
- 2% SDS
I) Submerge the membrane in stripping buffer: Incubate at 50°C for 30 minutes with occasional shaking. If more stringent conditions are needed, this incubate at 70°C.
IIa) Rinse the membrane under running water tap for 1-2 hours.
IIb) Wash the membrane twice for 10 minutes each, at room temperature, in 1x TBS, 0.05% Tween-20. Use a large volume (10-20 ml) of buffer for each wash.
III) Block the membrane for 1 hour at room temperature, or overnight at 4°C, in 1x TBS, 5% milk, 0.05% Tween-20.
0.5 L (sterile filter solution and keep at 4°C)
- 0.2 M Glycine, pH 2.5
- 0.05% Tween 20
I) Rinse blot off with 0.05% Tween 20 in PBS & put blot into a sealable bag cut to slightly bigger size than blot.
II) Add 5 to 10 ml stripping buffer & remove as much air as possible and seal bag.
III) Immerse into 80°C water bath and incubate for 20 min.
IV) Rinse blot several times with 0.05% Tween 20 in PBS & Block for 2 hr-overnight.
To make 1 Liter
- 15 g glycine
- 1 g SDS
- 10 ml Tween 20
- Volume up to 800ml dH2O
- pH to 2.2
- Volume to 1 L dH2O (<200ml)
- 10cm square tray @ 15ml of stripping buffer.
- (1st incubation) Incubate at room temperature 5-10 minutes. Discard buffer.
- (2nd incubation) @ 15ml of fresh stripping buffer. Incubate at room temperature 5-10 minutes. Discard buffer.
- Wash 2x10 minutes PBS
- Wash 2x5 minutes TBST
- Block. Proceed to 1ary incubation
"We have developed a guanidine hydrochloride-based (GnHCl) stripping solution (6M GnHCl, 0.2% Nonidet P-40 (NP-40), 0.1M β-mercaptoethanol, 20mM Tris-HCl, pH7.5) that can rapidly dissociate antibodies from immunoblots at room temperature without removing significant amounts of the transferred proteins. PMID: 19303392"
- 6M GnHCl
- 0.2% Nonidet P-40 (NP-40)
- 100mM β-mercaptoethanol
- 20mM Tris-HCl, pH7.5
- 2x30 min washes at room temperature for the Gly-HCl solution
- Wash 4 times with shaking (3 min each time) with 0.14M NaCl, 10mM Tris-HCl, pH7.2, (TBS) containing 0.05% NP-40 (TBSN) to remove the stripping solution.
The western blotting procedures are the same for PVDF or nitrocellulose, however the handling of these membranes are different prior to- and during- transfer of proteins from the SDS-PAGE gel to the membrane.
Nitrocellulose exhibits the highest sensitivity with very low backgrounds in all transfers, especially in protein blotting. Unlike PVDF, nitrocellulose wets out naturally, does not require methanol, and will not turn hydrophobic during the transfer process. Nitrocellulose is very easily blocked and does not need the many blocking steps required with PVDF.
Protocols for Western Blotting with PVDF and Nitrocellulose are the same with a few exceptions. PVDF is hydrophobic and therefore should be prewet in methanol before it is used. Wet the membrane in methanol for 15 seconds. Membrane should uniformly change from opaque to semi-transparent. Carefully place the membrane in ultrapure water and soak for 2 minutes. Then carefully place the membrane in transfer buffer and let equilibrate for at least 5 minutes.
Another change to note is that the SDS tolerances are not equivalent for PVDF and Nitrocellulose. The binding of protein to PVDF is much more sensitive to SDS levels. Too much SDS can inhibit the protein's ability to bind to the PVDF and can, in fact, help proteins already bound to the membrane to slip off. SDS levels should never exceed 0.05% for PVDF.