Thermo Scientific™ Restore™ Fluorescent Western Blot Stripping Buffer

Catalog No.	Quantity
PI62299	20 mL
PI62300	100 mL
FER62300X4	4 x 100 mL

Catalog No. PI62299 Supplier Thermo Scientific™ Supplier No. 62299

Description

Quickly and gently remove primary and near-infrared (IR) dye-labeled secondary antibodies from Western blots.

Thermo Scientific™ Restore Fluorescent Western Blot Stripping Buffer is a gentle and highly effective reagent for quickly removing primary and near-infrared (IR) dye-labeled secondary antibodies from Western blots.

Restore Fluorescent Western Blot Stripping Buffer enables the reuse of PVDF membranes, simplifying the Western blot optimization process and allowing the same blot to be reprobed with different primary antibodies to detect alternative targets. Restore Fluorescent Western Blot Stripping Buffer is for use with low-fluorescence PVDF membrane only (Part No. 22860).

Highlights:

• Fast – strip blots in only 15 minutes at room temperature

• Saves time – no need to run new gels and prepare a new blot

• Conserve samples – reprobe the same PVDF membrane for multiple targets

• Economical – less expensive than other commercially available stripping buffers

• Efficient – effectively strips blots the first time

Specifications

• Product Type: Western Blot Stripping Buffer
• Detection Method: Fluorescence
• Form: Liquid
• For Use With (Application): Western Blot
• Membrane Compatibility: PVDF
• Product Line: Restore
• Quantity: 20 mL
• Content And Storage: Upon receipt store at room temperature.

Frequently Asked Questions (FAQs)

How many times can I strip my blot using Restore or Restore Plus Western Blot Stripping Buffers ?

The stability of the attached (transferred and bound) protein will determine the number of times the membrane can be successfully re-probed after stripping. The protein may withstand stripping as many as four times or as few as one time.

What is the proper wash buffer to use after stripping with Restore or Restore Plus Western Blot Stripping buffers?

Wash the membrane with the same buffer as was used between antibody probing steps during the Western blotting procedure.

Can I strip membranes with Restore or Restore Plus Western Blot Stripping Buffer and reprobe for subsequent detection with fluorescent probes?

No. Although the fluorescent antibodies, like other antibodies, can be stripped with Restore Buffers, stripped membranes typically produce unacceptable background for subsequent fluorescent detection methods. We recommend Restore Fluorescent Western Blot Stripping Buffer, Cat. No. 62299 (20 mL) and Cat. No. 62300 (100 mL).

Note: Restore Fluorescent Western Blot Stripping Buffer is for use with low-fluorescence PVDF membrane (Cat. No. 22860) only.

Will the Restore and Restore Plus Western Blot Stripping Buffers remove precipitating substrates?

No. The antibodies are removed but the substrate leaves a permanent precipitate on the membrane that cannot be removed. Restore and Restore Plus Buffers are designed for procedures using chemiluminescent substrates. Please note that this is not compatible with fluorescence supstrates as it will result in increased background. For fluorescent substrates please use our Restore Fluorescent Western Blot Stripping Buffer. Please also see Tech Tip: Strip and Reprobe Western Blots (https://assets.thermofisher.com/TFS-Assets/BID/Technical-Notes/strip-reprobe-western-blots-tech-tip.pdf).

If I want to strip and re-probe a nitrocellulose membrane, what procedure do you recommend?

Thermo Fisher Scientific sells 4 ready-to-use buffers specifically intended for stripping Western blots prior to re-probing. Three of them can be used with both nitocellulose and PVDF membranes. We have Restore Western Blot Stripping Buffer (Cat. No. 21059), a trial size of this product (Cat. No. 21062), a product called Restore PLUS (Cat. No. 46428), which is formulated to strip off antibodies that are difficult to remove, and Restore Fluorescent Western Blot Stripping Buffer (Cat. No. 62299). The latter is used to strip fluorophore-labeled antibodies from PVDF membranes only.

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.

How do I strip my Western blot for reprobing with a different antibody?

Western blots can be stripped by incubation in Restore Western Blot Stripping Buffer at 37 degrees C for 5-15 min. If more stringent conditions are required, Restore Plus Western Blot Stripping Buffer can be used. Alternatively, membranes can be incubated in a stripping buffer consisting of 100 mM BME, 2% SDS, 62.5 mM Tris-HCL, pH 6.7, at 50 degrees C for 30 min with agitation. Wash the blot 3 times for 10 min each in PBST at room temperature. To reprobe with your antibody, the blot will need to be blocked again for 1 hr at room temperature.

Another stripping buffer is 0.1 M glycine-HCl (pH 2.5-3.0), the same solution commonly used for elution in immunoaffinity protocols. This solution will dissociate most antibody:antigen interactions at room temperature or 37 degrees C, but for strong antibody:antigen recognition, a 2 hr incubation may be necessary.

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