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TrueBlack® WB Blocking Buffer Kit

The TrueBlack® WB Blocking Buffer Kit is a ready-to-use buffer system that provides optimal specificity, sensitivity, and background signal suppression for fluorescence-based western blotting (WB).

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Product Description

The TrueBlack® WB Blocking Buffer Kit is a ready-to-use buffer system for fluorescence-based western blotting (WB). The buffers are designed to achieve optimal specificity and sensitivity by blocking background from the non-specific interaction between the fluorophore-labeled antibodies and the membrane.

  • Blocks non-specific protein binding, reducing background fluorescence over the entire membrane
  • Works as well or better than LI-COR’s Odyssey® Blocking Buffer, at a lower cost
  • Suppresses background caused by charged fluorescent dyes (performs better than BSA, gelatin, or casein)
  • Reduces antibody cross-reactivity, eliminating non-specific bands
  • Compatible with both polyvinylidene difluoride (PVDF) and nitrocellulose membranes
  • Can be used with antibodies labeled with all types of fluorophores spanning the visible and NIR spectrum
  • Contains non-mammalian blocking agents, for broad secondary antibody compatibility
  • TBS based buffer suitable for phosphoprotein detection
  • NOT designed for chemiluminescence detection using HRP conjugated antibodies

Non-specific signal in WB can arise from multiple sources, including antibody cross-reactivity with off-target proteins, non-specific antibody adsorption to the membrane, and membrane autofluorescence. Another potential cause of background that is not well-known is the effect of fluorescent dyes themselves on the specificity of labeled antibodies. Next-generation fluorescent dyes like Alexa Fluor® or CF® dyes often carry multiple negative charges to improve dye solubility and brightness of conjugates. However, the extra charge carried by the dye can result in non-specific antibody binding and background fluorescence. The TrueBlack® WB Blocking Buffer Kit blocks background from multiple sources including charged dye conjugates. It is especially advantageous for phosphoprotein detection, significantly improving specificity compared to conventional blocking buffers.

The TrueBlack® WB Blocking Buffer Kit belongs to our TrueBlack® line of background reducing agents for fluorescence applications, which includes TrueBlack® Lipofuscin Autofluorescence Quencher for tissue staining, and TrueBlack® IF Background Suppressor System (Permeabilizing) for blocking non-specific immunofluorescence staining.

When compared to LI-COR’s Odyssey® Blocking Buffer, the TrueBlack® WB Blocking Buffer works as well or better, and is priced lower on a per membrane basis. See below for a size comparison.

Comparison between TrueBlack® WB Blocking Buffer Kit and Odyssey® Blocking Buffer

ProductTrueBlack® WB Blocking
Buffer Kit
Odyssey® Blocking Buffer
Trial SizeFor 10 membranes125 mL for 4 membranes
Full SizeFor 50 membranes500 mL for 16 membranes
Number of membranes if Odyssey® Blocking Buffer is used for each blocking and antibody dilution step. TrueBlack® WB Blocking Buffer Kit includes enough buffers for all blocking and antibody incubation steps for the stated number of membranes.

 

Alexa Fluor is a registered trademark of Thermo Fisher Scientific. LI-COR and Odyssey are registered trademarks of LI-COR Inc.
 

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Support & Faq

Product shelf life, solubility, shipping and storage, and stability

Bioscience kits
The guaranteed shelf life from date of receipt for bioscience kits is listed on the product information sheet. Some kits have an expiration date printed on the kit box label, this is the guaranteed shelf life date calculated from the day that the product shipped from our facility. Kits often are functional for significantly longer than the guaranteed shelf life. If you have an older kit in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the kit still works for your application before processing a large number of samples or precious samples.

Antibodies and other conjugates
The guaranteed shelf life from date of receipt for antibodies and conjugates is listed on the product information sheet. Antibodies and other conjugates often are functional for significantly longer than the guaranteed shelf life. If you have an older conjugate in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

For lyophilized antibodies, we recommend reconstituting the antibody with glycerol and antimicrobial preservative like sodium azide for the longest shelf life (note that sodium azide is not compatible with HRP-conjugates).

Chemicals, dyes, and gel stains
Biotium guarantees the stability of chemicals, dyes, and gel stains for at least a year from the date you receive the product. However, the majority of these products are highly stable for many years, as long as they are stored as recommended. Storage conditions can be found on the product information sheet or product safety and data sheet, material safety data sheet, and on the product label. Fluorescent compounds should be protected from light for long term storage.

If you have a Biotium compound that has been in storage for longer than one year that you wish to use, we recommend performing a small scale positive control experiment to confirm that the compound still works for your application before processing a large number of samples or precious samples.

Expiration date based on date of manufacture (DOM)
If your institution requires you to document expiration date based on date of manufacture for reagents, please contact techsupport@biotium.com for assistance.

Chemical products with special stability considerations:

Esters

Ester compounds include the following:
• Succinimidyl esters (SE, also known as NHS esters), such as our amine-reactive dyes
• Acetoxymethyl esters (AM esters) such as our membrane-permeable ion indicator dyes
• Diacetate-modified dyes, like ViaFluor™ 405, CFDA, and CFDA-SE cell viability/cell proliferation dyes

Ester dyes are stable in solid form as long as they are protected from light and moisture. Esters are not stable in aqueous solution. Concentrated stock solutions should be prepared in anhydrous DMSO (see Biotium catalog no. 90082). Stock solutions in anhydrous DMSO can be stored desiccated at -20°C for one month or longer. Esters should be diluted in aqueous solution immediately before use. Succinimidyl esters (SE) should be dissolved in a solution that is free of amine-containing compounds like Tris, glycine, or protein, which will react with the SE functional group. AM esters and diacetate compounds should be dissolved in a solution that is free of serum, because serum could contain esterases that would hydrolyze the compound.

A note on CF™ dye succinimidyl ester stability
Succinimidyl esters are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Heavily sulfonated dyes, such as the Alexa Fluor® dyes, DyLight® dyes and IRDyes® are particularly hygroscopic, worsening the hydrolysis problem. For example, the percent of active Alexa Fluor® 488 succinimidyl ester (SE) could be well below 50% by the time of application (according to the manufacturer’s product datasheet). In a number of Alexa Fluor® SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while in all of Biotium’s CF™ dyes the SE group is prepared from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF™ dye SE reactive groups to hydrolysis, resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes.

Maleimides, MTS and thiosulfate dyes
Like the succinimidyl ester dyes, these dyes are also susceptible to hydrolysis, although generally to a much lower degree. Thus, for long term storage, anhydrous DMSO is recommended for making stock solutions.

Other reactive dyes
Amines, aminooxy (also known as oxylamine), hydrazide, azide, alkyne, BCN, and tyramide reactive dyes, as well as dye free acids, are generally stable in aqueous solution when stored at -20°C for 6-12 months or longer, as long as no compounds are present that may react with the dye’s functional group. See the product information sheets for specific reactive dyes more information.

Coelenterazines and D-luciferin

Coelenterazines are stable in solid form when stored as recommended; they are not stable in aqueous solution. Concentrated coelenterazine stock solutions (typically 1-100 mg/mL) should be prepared in ethanol or methanol; do not use DMSO or DMF to dissolve coelenterazines, because these solvents will oxidize the compounds. Ethanol or methanol stocks of coelenterazine can be stored at -20°C or below for six months or longer; alcohol stocks may evaporate during storage, so use tightly sealing screw cap vials and wrap the vials with Parafilm for long term storage. Propylene glycol also can be used as a solvent to minimize evaporation. If the solvent evaporates, the coelenterazine will still be present in the vial, so note the volume in the vial prior to storage so that you can adjust the solvent volume to correct for evaporation if needed. Prepare working solutions in aqueous buffers immediately before use. Coelenterazines are stable for up to five hours in aqueous solution.

Aquaphile™ coelenterazines are water soluble formulations of coelenterazines. They are stable in solid form when stored as recommended. Aquaphile™ coelenterazines should be dissolved in aqueous solution immediately before use. They are stable for up to five hours in aqueous solution.

Note that coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

D-luciferin is stable in solid form and as a concentrated stock solution when stored as recommended; it is not stable at dilute working concentrations in aqueous solution. Prepare concentrated D-luciferin stock solutions (typically 1-100 mg/mL) in water, and store in aliquots at -20°C or below for six months or longer. Prepare working solutions immediately before use.

Category: Mix-n-Stain™ Antibody Labeling Kits, DNA Quantitation Kits, NucView® Caspase 3 Enzyme Substrates, Luciferase Assays, CF® Dyes, Product shelf life, solubility, shipping and storage, and stability

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Many of our solid compounds are packaged by lyophilization, in which case they usually do not appear as fluffy powders, but form a film or coating on the sides of the vial. Simply add the appropriate volume of the recommended solvent to the vial to make the desired concentration stock solution, and swirl or gently vortex to mix. Make sure the solvent comes in contact with the inside walls of the vial to fully recover the product.

Category: Product shelf life, solubility, shipping and storage, and stability

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Most of our products are stable at room temperature for many days, but we recommend storage at 4°C or -20°C to prolong shelf life. In the case of many of our aqueous dye solutions, the compounds are very stable at room temperature, but we recommend cold storage to prevent the growth of mold or other microbes over time. Therefore, to save on shipping costs, products with recommended storage at 4°C or -20°C may ship at ambient temperature or with an ice pack. These products may thaw without affecting product performance. When you receive the product, place it under the recommended storage conditions.

Category: Mix-n-Stain™ Antibody Labeling Kits, DNA Quantitation Kits, NucView® Caspase 3 Enzyme Substrates, Luciferase Assays, CF® Dyes, Product shelf life, solubility, shipping and storage, and stability

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Shipping/Shelf-life

Shipping & Handling Disclaimer: Shipping and handling methods are calculated based on item temperature storage recommendation. Large, multi-item orders may be assessed additional fees. Shipping to Puerto Rico may be assessed additional fees. A Biotium customer service representative will contact you if additional fees are required. For expedited shipping, please request in the Order Notes section of the checkout page. Please note that products with recommended storage at 4°C or -20°C may ship at ambient temperature. This will not affect product performance. When you receive the product, place it under the recommended storage conditions.