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TrueBlack® IF Background Suppressor System (Permeabilizing)

The TrueBlack® Background Suppressor System is designed for optimal blocking of non-specific antibody binding as well as direct interaction of fluorescent dyes on antibodies with cells or tissue sections.

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Product Description

Features

  • Suppresses background from non-specific antibody binding and charged fluorescent dyes
  • More efficient than Image-iT® FX, block & permeabilize in just 10 minutes
  • Complete system for blocking, permeabilizing, and antibody dilution
  • Non-mammalian blocking agents, for broad secondary antibody compatibility
  • For staining of cells or tissue sections

Components

  • TrueBlack® IF Background Suppressor
    Blocks background from non-specific binding of proteins & charged dyes
  • TrueBlack® IF Blocking Buffer
    Excellent general blocking agent & antibody diluent

The TrueBlack® Background Suppressor System is a buffer system designed for optimal blocking of non-specific staining for immunofluorescence (IF). The buffers are designed to block background from both non-specific antibody binding as well as direct interaction of fluorescent dyes on antibodies with cells or tissue sections.

Non-specific signal in immunofluorescence can arise from multiple sources, including antibody cross-reactivity with off-target proteins, non-specific antibody adsorption to the sample, and 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, which can affect signal-to-noise of immunostaining, particularly for low abundance targets. While conventional blocking agents like BSA, gelatin, or casein can reduce non-specific protein binding, they are not effective at blocking background from charged dyes.

TrueBlack® Background Suppressor is designed for blocking both non-specific protein binding as well as background from charged dyes. The buffers can be used for both blocking and antibody dilution. For some antibodies, best results are obtained when the Background Suppressor is used for blocking and the Blocking Buffer is used for antibody dilution. The Blocking Buffer alone also can provide excellent results when used for both blocking and antibody dilution for conjugates that do not carry excess charge. We recommend testing the different buffers to find the combination that works best for your antibody. Examples of charged dyes that show improved signal to noise with the Background Suppressor are CF®405S, CF®405M, Alexa Fluor® 647, and Cy®5.5

The buffers in the TrueBlack® Background Suppressor System (Permeabilizing) contain non-mammalian based blocking agents plus detergent for simultaneous blocking and permeabilization for intracellular immunofluorescence. The Background Suppressor contains additional blocking agents for suppressing background from charged dyes. The buffers are provided in dropper bottles for easy dispensing.

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

Alexa Fluor and Image-iT are registered trademarks of Thermo Fisher Scientific; Cy Dye is a registered trademark of GE Healthcare.

 

 

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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.