Thermo Scientific™ Restore™ PLUS Western Blot Stripping Buffer

Catalog No.	Quantity
FER46430X4	4 x 500 mL
PI46428	30 mL
PI46430	500 mL

Catalog No. FER46430X4 Supplier Thermo Scientific™ Supplier No. 46430X4

Description

Remove high-affinity antibodies from membranes so they can be reprobed and detected with chemiluminescent substrates.

Thermo Scientific Restore PLUS Western Blot Stripping Buffer is an advanced formula for removing bound primary and secondary antibodies from membranes so they can be reprobed and detected with chemiluminescent substrates.

Features of Restore PLUS Western Blot Stripping Buffer:

• Ready and easy to use—no dilution necessary; no offensive odors; store at room temperature
• Compatible—use on nitrocellulose and PVDF membranes, whether still wet or already dry; works with practically any blocking buffer, enzyme conjugate and chemiluminescent substrate
• Cost effective—save valuable time and samples; strip blots effectively the first time
• Robust yet gentle—transferred proteins remain viable; strip the same blot up to five times
• Flexible—strip and reprobe to optimize antibody concentrations or to detect a new antigen with different antibodies

Restore PLUS Buffer is an alternative formulation of the original Thermo Scientific Restore Western Blot Stripping Buffer. Restore PLUS Stripping Buffer was designed for use with antibodies that are difficult to remove from western blots and require longer incubation times or incubation temperatures greater than 22°C with gentler formulations. High-affinity antibodies can be quickly and effectively stripped from western blots at room temperature without removing transferred proteins, thereby allowing multiple reprobes of the target.

Protocol Summary:

• Wash blot to remove chemiluminescent substrate
• Incubate blot in Restore Western Blot Stripping Buffer for 5 to 15 minutes at room temperature
  • (or incubate at 37°C for high affinity antibodies)
• Remove blot and wash in Wash Buffer
• Block membrane
• Test for sufficient removal of antibodies
• Perform next immunoblot experiment

Specifications

• Form: Liquid
• Product Line: Restore
• Quantity: 4 x 500 mL
• Content And Storage: Upon receipt store at room temperature. Product shipped at ambient temperature.

Frequently Asked Questions (FAQs)

How can I store, strip, and reuse my western blot?

For nitrocellulose or PVDF membrane following Western blot detection using a chemiluminescent or fluorescent substrate system: Following transfer, air dry the membrane and place in an envelope, preferably on top of a supported surface to keep the membrane flat. The blot can be stored indefinitely at -80 degrees C. When ready to reprobe, prewet the PVDF blot with alcohol for a few seconds, followed by a few rinses with pure water to reduce the alcohol concentration. Then proceed as normal with blocking step.

FOR STRIPPING/REPROBING OF MEMBRANES: Harsh protocol (see NOTE below for modifications)

1) Submerge the membrane in stripping buffer (100 mM BME, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) and incubate at 50 degrees C for 30 min with occasional agitation. If more stringent conditions necessary, incubate at 70 degrees C.

2) Wash 2 x 10 min in TBS-T/PBS-T at room temperature.

3) Block the membrane by immersing in 5% blocking reagent TBS-T or PBS-T for 1 hr at room temperature.

4) Immunodetection

NOTE: Often you don't need such harsh conditions to remove antibodies from their proteins. The stringency of one or several of the variables can be decreased: lower the temperature, decrease the time, less BME, less SDS, etc. An especially mild but still often effective stripping protocol is lower pH incubation. Example: pH 2.0 Tris 50-100 mM, 30-60 min incubation (you may do two incubations if you wish). Then rinse and block as usual. If you do not wish to re-use the membrane immediately after stripping, you can store the membrane in plastic wrap (wet, you do not want it to dry out). Another simple, mild stripping buffer is 0.1 M glycine•HCl (pH 2.5-3.0), incubation 30 min to 2 hrs room temperature or 37 degrees C, depending on the antibody.

For Research Use Only. Not for use in diagnostic procedures.