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

Answer

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.

Answer Id:: E5217

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Products for Western Blotting Product Literature

Can I use eBioscience antibodies for immunoblotting (WB)? Product FAQ

Answer

We do not routinely test our antibodies in house by western blotting (WB) unless it was specifically developed for that purpose; therefore, we rely on the literature as the basis for reporting WB as an approved application for a given clone. If a product is suitable for WB, it will be noted under reported applications on the products technical data sheet. Please refer to the Product Technical Datasheet for more information.

Answer Id:: E14508

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Can I use fluorescent secondary antibodies for western blots? Product FAQ

Answer

Yes, as long as the instrument used to analyze the blots provides the appropriate excitation wavelengths and emission filter sets to detect fluorescence of the selected dye(s). The limits of detection are dependent upon the amount of protein/peptide per spot/band, using the correct excitation and emission settings for the dye-conjugated antibody, and the sensitivity of the camera.
Another alternative to dye-conjugated secondary antibodies are the quantum dot-conjugated antibodies provided in our WesternDot products. For more information: https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/detect-proteins-western-blot/western-blot-detection-reagents/fluorescent-western-blot-detection/westerndot-fluorescent-immunodetection-kits.html

Answer Id:: E18039

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Has the BenchPro 4100 Card Processing Station been discontinued? Product FAQ

Answer

Yes, the BenchPro 4100 Card Processing Station has been discontinued as of December 31, 2014. The current BenchPro 4100 Western Cards (https://www.thermofisher.com/order/catalog/product/WP1001) and Reagent Vials (https://www.thermofisher.com/order/catalog/product/WP3001) will continue to be available for purchase. We offer the new iBind Western Device (http://www.thermofisher.com/us/en/home/life-science/protein-expression-and-analysis/western-blotting/western-blot-detection/ibind-western-system.html) as an alternative system for automated western blot processing.

Answer Id:: E10882

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Highly specific confirmatory western blot test for African swine fever virus antibody detection using the recombinant virus protein p54. Citations & References

  • Authors: Alcaraz C; Rodriguez F; Oviedo JM; Eiras A; De Diego M; Alonso C; Escribano JM
  • Journal: Journal of Virological Methods
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What applications are appropriate for your Anti-Xpress antibody, Anti-V5 antibody, Anti-HisG antibody, and Anti-myc antibody? Product FAQ

Answer

ELISA, Western blot, Immunoprecipitation, Immunofluorescence, and Immunohistochemistry protocols have all been used successfully with our epitope tag antibodies. Please consult the product manuals for specific protocols and dilutions.

Answer Id:: E3641

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User Guide: Pierce ECL Plus Western Blotting Substrate Manual / Product Insert

  • Version: B.0
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What is the difference between Western Blot Signal Enhancer and SuperSignal Western Blot Enhancer? Is there any benefit to one versus the other? Product FAQ

Answer

The reagents in both kits are different. Western Blot Signal Enhancer (Cat. No. 21050) is a 2-step pre-treatment that occurs before blocking. It basically relaxes the protein on the membrane making the epitope easier to detect. SuperSignal Western Blot Enhancer (Cat. Nos. 46640, 46641) contains a membrane treatment reagent and a primary antibody diluent that increase both signal intensity and sensitivity 3- to 10-fold compared to a detecion performed without it. We have found that SuperSignal Western Blot Enhancer shows much less background while enhancing signal and sensitivity when compared to Western Blot Signal Enhancer.

Answer Id:: E15178

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What is a suitable generic antibody starting concentration for Western blotting (WB), ELISA, immunohistochemistry (IHC), immunofluorescence (IF), immunoprecipitation (IP), or flow cytometry (FC)? Product FAQ

Answer

It is recommended that you first check the product insert for any recommended usage guidelines.

For general purposes, starting concentrations are as follows:
ELISA--0.5-1.0 µg/mL
WB--1-5 µg/mL
IP--2-5 µg per reaction (1 X 10e6 cells)
IHC/IF--1-10 µg/mL
FC--10-20 µg/mL

Please note that these are general guidelines only.

Answer Id:: E4942

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Do I have to use a SuperSignal Western Blot substrate after using the SuperSignal Western Blot Enhancer? Product FAQ

Answer

Not necessarily. The SuperSignal Western Blot Enhancer will work with any chemiluminescent substrate (including SuperSignal Western Blot substrates), as well as colorimetric or fluorometric detection systems.

Answer Id:: E15179

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

Answer

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.

Answer Id:: E3862

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When should I use the SuperSignal Western Blot Enhancer (Cat. Nos. 46640 and 46641)? Product FAQ

Answer

The SuperSignal Western Blot Enhancer kit contains two reagents:

- Antigen Pretreatment Solution: used after transferring proteins to the membrane and before blocking or staining the membrane
- The Primary Antibody Diluent: used to dilute the primary antibody

Answer Id:: E15177

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User Guide: SuperSignal Western Blot Enhancer Manual / Product Insert

  • Version: Jan. 2015
Catalog #

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

Answer

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).

Answer Id:: E8468

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