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WESTERN BLOTTING & PROTEIN ANALYSIS

Reagents for Western, SDS-PAGE, & Protein Thermal Stability

Biotium offers innovative solutions for protein detection and analysis. Jump to a section below to learn more:

For superior signal to noise in fluorescent western
Antibody conjugates of our best-in class NIR dyes
Super-sensitive & linear near-IR protein quantitation in gels or blots
Fast, easy & non-toxic One-Step Stains
GloMelt™ is highly sensitive & tolerant of detergent
AccuOrange™ highly sensitive fluorescence protein assay
Prestained protein markers, buffers, & detergents

TrueBlack® WB Blocking Buffer Kit

The TrueBlack® WB Blocking Buffer Kit is a ready-to-use buffer system for fluorescence-based western blotting (WB). The buffers yield optimal specificity and sensitivity by blocking non-specific interactions of dye-labeled antibodies with proteins and the blotting membrane.

TrueBlack® WB Blocking Buffer Kit Features

  • Blocks as well or better than Odyssey® Blocking Buffer, at a lower price
  • Reduces non-specific protein bands and background over entire membrane
  • Suppresses background from charged dyes better than BSA, gelatin, or casein
  • Compatible with PVDF and nitrocellulose membranes
  • Contains no mammalian proteins, for broad antibody compatibility
  • For visible and near-IR fluorescent westerns
Figure 1. Western detection of phospho-Erk1/2 in PDGF-stimulated NIH-3T3 cell lysate. Membranes were blocked with fish gelatin blocking buffer, LI-COR® Odyssey® TBS Blocking Buffer, or TrueBlack® WB Blocking Buffer. Phosphorylated Erk was detected using rabbit anti-pErk1/2 and CF®680R donkey anti-rabbit antibodies. TrueBlack® WB Blocking Buffer gave lower background fluorescence and better specificity compared to the other buffers.

Superior western blocking for next-generation fluorescent dyes

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 is the effect of fluorescent dyes themselves on the specificity of labeled antibodies. Highly charged dyes like Alexa Fluor® or CF® dyes have improved solubility and brightness of conjugates compared to uncharged dyes. However, the extra charge carried by antibodies labeled with these dyes can result in non-specific binding to proteins and membranes. The TrueBlack® WB Blocking Buffer Kit blocks background from multiple sources including charged dye conjugates (Figure 2). TrueBlack® blocking buffer is especially advantageous for phosphoprotein detection, significantly improving specificity compared to conventional blocking buffers (Figure 1).

Switch from Odyssey® Blocking Buffer and save

TrueBlack® WB Blocking Buffer performs as well or better for fluorescent WB compared to LI-COR’s Odyssey® Blocking Buffer (Figure 1), and is priced lower on a per membrane basis.

 

Figure 2. Western detection of tubulin in HeLa cell lysate with mouse anti-tubulin and Alexa Fluor® 790 goat anti-mouse antibodies. Membranes were blocked with fish gelatin blocking buffer or TrueBlack® WB Blocking Buffer. The highly negatively charged Alexa Fluor® 790 labeled antibody showed non-specific binding to the PVDF membrane and cellular proteins, which was blocked by TrueBlack® WB Blocking Buffer. Lanes 1-3: 10 ug, 1 ug, or 0.1 ug HeLa cell total protein. Note: Alexa Fluor ®790 dye was used to demonstrate non-specific background from highly charged dyes. Biotium’s near-IR CF® dyes are peggylated, a structural feature that renders the dyes highly water soluble without excessive negative charge, resulting in significantly lower background. Learn more about Near-IR CF® Dyes.

Compare TrueBlack® WB Blocking Buffer Kit with 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.

LI-COR and Odyssey are registered trademarks of LI-COR Inc.

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Near-IR CF® Dye conjugates for western blotting

Near-infrared (near-IR) western detection is highly sensitive, and offers advantages of wider linear range and multiplexing capability compared to chemiluminscence detection (see our webinar to learn more). Biotium’s near-IR CF® dyes are the brightest and most photostable available. Learn more about CF®680 and CF®770 dyes for near-IR western, In Cell Western®, and other applications.

We offer wide selection of primary and secondary antibodies conjugated to our exceptional near-IR CF® dyes for western blot. We also offer HRP conjugates for chemiluminescence detection.

CF® dye conjugates are brighter than IRDye® conjugates for near-infrared western blotting. A dilution series of HeLa cell lysate (from 2 ug to 0.125 ug) was transferred to PVDF membranes. Mouse anti-tubulin and rabbit anti-COX IV antibodies were detected using CF®680 anti-mouse and CF®770 anti-rabbit, or the corresponding IRDye® conjugates, and scanned on LI-COR® Odyssey®. Band quantitation showed ~50% brighter signal with CF® dyes. M: marker.
CF®790 has fewer negative charges than Alexa Fluor® 790, resulting in more specific antibody conjugates. Western blots of HeLa lysate (two dilutions, lanes 1 and 2) were probed with mouse anti-tubulin antibody followed by goat anti-mouse conjugates of the indicated dyes. The Alexa Fluor® 790 conjugate shows multiple non-specific bands compared to CF®790. M: molecular weight marker.

Near-IR CF Dye Secondary Antibodies for Multiplex WB

ConjugateCross-adsorptionCF®680 (681/698 nm)CF®680R (680/701 nm)CF®750 (755/777 nm)CF®770 (770/797 nm)CF®790 (784/806 nm)
Donkey Anti-GoatCk, GP, Hs, Hu, Ms, Rb, Rt, SHm20060201962036220277
Donkey Anti-Guinea PigBv, Ck, Gt, Hs, Hu, Ms, Rb, Rt, Shp, SHm2024120242
Donkey Anti-MouseBv, Ck, Gt, GP, Hs, Hu, Rb, Shp, SHm2019420363
Donkey Anti-RabbitBv, Ck, Gt, GP, Hs, Hu, Ms, Rt, Shp, SHm2041820195202982048420344
Donkey Anti-SheepCk, GP, Hs, Hu, Ms, Rb, Rt, SHm20062
Goat Anti-Guinea PigBv, Ck, Gt, Hs, Hu, Ms, Rb, Rt, SHm, Shp20497204962049920498
Goat Anti-MouseBv, Hs, Hu, Rb, Sw2006520192204632007720342
Goat Anti-Mouse IgG1Bv, Hu, Rb2025320254
Goat Anti-Mouse IgG2aBv, Hu, Rb202632084220264
Goat Anti-Mouse IgG2bBv, Hu, Rb202732043020274
Goat Anti-RabbitHu, Ms, Rt20067201932007820343
Goat Anti-RatBv, Hs, Hu, Rb2006920383
Rabbit Anti-MouseHu20061
Don’t see what you’re looking for? Contact us! We may be able to add a new conjugate to our catalog, or perform a custom labeling for you.
Bv: bovine, Ck: chicken, GP: guinea pig, Gt: goat, Hs: horse, Hu: human, Ms: mouse, Rb: rabbit, Rt: rat, SHm: Syrian hamster, Shp: sheep, Sw: swine

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One-Step Blue® & One-Step Lumitein™ Protein Gel Stains

Our One-Step protein gel stains are low-cost, quick, and easy-to-use. Simply add stain to your SDS-PAGE gel without fixing or washing, and stain for 5 to 60 min. These stains are non-toxic for safer handling, and are certified under CCR Title 22 as non-hazardous for drain disposal after pH neutralization. One-Step Blue® replaces Coomassie® for visible staining or near-IR fluorescence. One-Step™ Lumitein replaces SYPRO® Ruby for UV box or gel scanner, and One-Step™ Lumitein UV replaces Oriole™ for UV transilluminator.

One-Step Stains Features

  • Fast, simple staining
  • No fixing, microwaving, or washing
  • Aqueous-based and non-toxic for drain disposal

One-Step Blue® is an alternative to time consuming Coomassie® staining, for fast and easy visible staining (left) or near-infrared imaging in the LI-COR® Odyssey® 800 channel (right).
Red fluorescent One-Step Lumitein™ is an alternative to time-consuming and expensive SYPRO® Ruby protein gel stain, and it can be imaged on a UV box or fluorescence gel scanner
Red fluorescent One-Step Lumitein™ UV Protein Gel Stain is designed for imaging using a UV transilluminator. It is an alternative to Oriole™ Gel Stain, but does not contain hazardous solvents and does not cause gel shrinkage.

Product Information

ProductReplacement forCatalog numberUnit sizeProduct protocolSafety report
One-Step Blue®
Coomassie stain21003-1L1LPI-21003
One-Step Gel Stains Safety Report
One-Step Lumitein™SYPRO® Ruby Protein Gel Stain21004-1L1LPI-21004
21004-4L4L
One-Step Lumitein™ UVOriole™ and Flamingo™ Protein Gel Stains21005-1L1LPI-21005
21005-4L4L

Lumitein and its related technologies are covered by US and international patents. SYPRO is a registered trademark of Molecular Probes, Inc. Oriole is a trademark of Bio-Rad Laboratories. Coomassie is a registered trademark of Imperial Chemical Industries.

 

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Mix-n-Stain™ Near-IR Total Protein Prestain Kits

Mix-n-Stain™ Total Protein Prestain Kits allow simple, sensitive and highly linear protein quantitation on SDS-PAGE gels and western blot membranes. The kits allow you to label purified proteins or cell lysates with our near-infrared CF® dyes before running the samples on SDS-PAGE. After electrophoresis, the bands can be visualized using a fluorescent gel scanner, allowing detection of as little as 1 ng protein per band. Labeled proteins also can be transferred to membranes for western blotting. The staining demonstrates excellent linearity for quantitation of total protein over a wide dynamic range, outperforming traditional western blot normalization based on housekeeping protein detection.

Mix-n-Stain™ Total Protein Prestain Features

  • Superior linearity for western normalization compared to housekeeping proteins
  • Highly sensitive protein quantitation on PAGE gels or western membranes
  • Fast and simple labeling of proteins or lysates, no purification required
  • Detect as little as 1 ng protein in gel

In-gel fluorescence image of bovine serum albumin (BSA) labeled with the CF®680T total protein prestaining kit on SDS-PAGE gel. Protein content for each lane ranges from 10 ug to 1 ng, from left to right. The bands above and below the major bands are from impurity proteins in the BSA sample. The excess dye runs to the very bottom of the gel. Inset: part of the image with enhanced brightness to visualize the bands with 10, 5, and 1 ng of BSA.
Mix-n-Stain™ total protein prestaining kits for western blot normalization. HeLa cell lysate in serial dilution was labeled with (A) the CF®770T total protein prestain before SDS-PAGE and transfer to PVDF, or (B) mouse anti-tubulin primary antibody and CF®770T goat anti-mouse secondary antibody after transfer to PVDF. (C) Plots of band intensity vs protein content for CF®770T labeled lysate (shown in A), CF®680T labeled lysate (membrane not shown), and tubulin WB (shown in B). The Mix-n-Stain™ total protein prestaining kits show better linearity compared to housekeeping protein detection.

Product Information

ProductCatalog numberSizeAbs/EmImaging Systems / Detection channels
Mix-n-Stain™ CF®680T Total Protein Prestain Kit92400-T200 labelings681 / 698 nmAmersham Typhoon™ Trio Cy®5 channel
Amersham Typhoon™ 5 IR short channel
LI-COR® Odyssey® 700 channel
924001000 labelings
Mix-n-Stain™ CF®770T Total Protein Prestain Kit92401-T200 labelings764 / 787 nmAmersham Typhoon™ 5 IR long channel
LI-COR® Odyssey® 800 channel
924011000 labelings

Typhoon is a trademark and Cy dye is a registered trademark of GE Healthcare; Odyssey is a registered trademark of LI-COR Biosciences.

 

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GloMelt™ Protein Thermal Stability Assay

Thermal shift assay for protein stability

The thermal shift assay, also called Protein Thermal Shift™, differential scanning fluorimetry, or Thermofluor assay, is a simple technique for measuring protein denaturation temperature. It can be used to screen conditions that affect protein thermal stability, such as mutations, ligand binding, and buffer formulations. The assay is rapid and is performed on a quantitative PCR system. Thermal shift is compatible with high throughput screening and requires much less protein than methods like circular dichroism and differential scanning calorimetry.

Environmentally sensitive fluorescent dyes can be used to monitor the temperature dependent unfolding of a protein. The protein’s melting temperature (Tm) is a reporter of the protein’s thermal stability.

GloMelt™ Dye for protein thermal stability

GloMelt™ dye undergoes fluorescence enhancement upon binding to hydrophobic regions of denatured proteins, and can be used to detect protein unfolding in thermal shift assay. GloMelt™ dye is optimized for detection in the SYBR® Green channel, so it generates much stronger signal than SYPRO® Orange and PROTEOSTAT® TS dyes, which have spectra that are not well-matched to common qPCR fluorescence channels. Because GloMelt™ has green fluorescence, it also can be used with ROX normalization for improved replicate consistency.

GloMelt™ tolerates a wide variety of common stabilizers and destabilizers, and unlike SYPRO® Orange, it performs well in the presence of detergents. SYPRO® Orange and PROTEOSTAT® TS dyes are highly hydrophobic, so after dilution in buffer the dyes must be used immediately. In contrast, GloMelt™ has excellent water solubility, allowing storage of diluted dye solutions for greater convenience and less wasted dye.

GloMelt™ Features

  • Green fluorescence optimal for qPCR instruments and ROX normalization
  • Compatible with high detergent concentrations
  • Compatible with wide pH range, reducing agents, and common buffers/excipients
  • Great for high throughput assays, low reaction volumes and low protein concentrations
  • Highly soluble and stable in aqueous buffers

GloMelt™ has much better detergent tolerance than SYPRO® Orange. IgG melt curve plots in the presence of detergent. A thermal shift assay was performed on 20 ug IgG in the presence of 5X SYPRO® Orange or 1X GloMelt™ dye, using a QuantStudio™ 5 qPCR system. The presence of detergent inhibited the SYPRO® Orange assay, but had little affect on the GloMelt™ curve.
GloMelt™ tolerates reducing agents, unlike PROTEOSTAT® TS. IgG melt curve plots in the presence of DTT. A thermal shift assay was performed on 25 ug IgG in the presence of 1X PROTEOSTAT® TS dye or 1X GloMelt™ dye, using a QuantStudio™ 5 qPCR system. The presence of DTT drastically reduced the sensitivity of the PROTEOSTAT® assay, but had little affect on the GloMelt™ dye. As expected, DTT reduced IgG thermal stability.
Normalization of GloMelt™ signal to ROX reference dye can improve results by increasing replicate consistency. A thermal shift assay was performed on 20 ug IgG in the presence 1X GloMelt™ dye and 50 nM ROX. After ROX normalization the standard deviation was reduced more than 5-fold.

Product Information

ProductKit contentsCatalog numberSize*
GloMelt™ Kit• GloMelt™ Dye
• Goat IgG Control
33021-T200 reactions
33021-12000 reactions
GloMelt™ Kit with ROX• GloMelt™ Dye
• Goat IgG Control
• ROX Reference Dye
33022-T200 reactions
33022-12000 reactions
* Size based on 20 uL reaction volume

SYPRO is a registered trademark of Thermo Fisher Scientific; Protein Thermal Shift and QuantStudio are trademarks of Thermo Fisher Scientific; PROTEOSTAT is a registered trademark of Enzo Life Sciences.

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AccuOrange™ Fluorescent Protein Quantitation Assay

AccuOrange™ Protein Quantitation Kit is a highly sensitive fluorescence-based assay for quantitating purified protein or antibody samples in 96-well format. The detection range of the assay is 0.1-15 ug/mL protein. AccuOrange™ is much more sensitive than traditional protein quantitation assays such as BCA, Bradford and Lowry, and is more linearity and reproducible compared to the NanoOrange® protein quantitation assay. The assay shows minimal variability between different proteins, and has stable fluorescence signal for up to 16 hours. The AccuOrange™ assay has low tolerance for detergents, and is not recommended for use with cell lysates.

The AccuOrange™ fluorescent protein quantitation assay is more  linear and less variable than the NanoOrange® assay.
AssayDetection RangeComments
AccuOrange™0.1-15 ug/mLFluorescence-based (480/598 nm)
Highly linear
Signal stable for at least 16 hours
Compatible with reducing agents
Not compatible with detergents
NanoOrange®0.1-10 ug/mLFluorescence-based (470/570 nm)
Non-linear
Fluorescence stable for 6 hours
Compatible with reducing agents
Not compatible with detergents
Modified Lowry1-1500 ug/mLAbsorbance-based (750 nm)
Non-linear
Not compatible with reducing agents or detergents
BCA20-2000 ug/mLAbsorbance-based (562 nm)
Highly linear
Signal not stable over time
Not compatible with reducing agents
Compatible with detergents
Bradford (Coomassie®)50-500 ug/mLAbsorbance-based (595 nm)
Signal not stable over time
Non-linear
Compatible with reducing agents
Not compatible with detergents
Pierce® 660 nm50-2000 ug/mLAbsorbance-based (660 nm)
Non-linear
Compatible with reducing agents & detergents
A28050-2000 ug/mLAbsorbance-based (280 nm)
High protein-protein variability
Contaminants and detergents can affect results

Product Information

ProductCatalog numberSizeFeatures
AccuOrange™ Protein Quantitation Kit30071-T200 assays• Highly sensitive: detect 0.1-15 ug/mL protein
• Excellent linearity and low variability
• Stable fluorescence signal
• Compatible with reducing agents and other additivies
• For use with purified protein or antibody samples
300711000 assays

NanoOrange is a registered trademark and Pierce is a trademark of Thermo Fisher Scientific; Coomassie is a registered trademark of Imperial Chemical Industries.

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Accessory Reagents for Western Blotting

See our selection of prestained markers, buffers, blocking agents, detergents, and other reagents and accessories below.

Product Information

ProductCatalog No.SizeFeatures
Peacock™ Prestained Protein Marker2153050 uL or 500 uL• Visible color protein marker for SDS-PAGE and western
• 8 blue bands ranging from 10 kDa to 180 kDa
• Red/green bands at 75 kDa/25 kDa
Peacock™ Plus Prestained Protein Marker2153150 uL or 500 uL• Visible color protein marker for SDS-PAGE and western
• 10 blue bands ranging from 8 kDa to 245 kDa
• Red/green bands at 75 kDa/25 kDa
Ponceau S Solution220011 L• Stain proteins on PVDF or nitrocellulose membranes with visible pink dye
• Fast & reversible visualization of protein transfer before western detection
TrueBlack® WB Blocking Buffer Kit23013For 10 membranes or 50 membranes• Superior blocking for fluorescent WB
• Works as well or better than Odyssey® Blocking Buffer, at a lower cost
• Suppresses background caused by charged fluorescent dyes
• Reduces antibody cross-reactivity, eliminating non-specific bands
10X Fish Gelatin Blocking Agent22010100 mL• Provides excellent blocking for IF or western
• Add to buffer of your choice (PBS or TBS)
• Compatible with anti-goat and anti-sheep secondaries
Fish Gelatin Powder220112 x 50 g• Gelatin from cold water fish skin for blocking for IF or western
• Compatible with anti-goat and anti-sheep secondaries
Bovine Serum Albumin, 30% Solution22014100 mL• Commonly used blocking agent and antibody or protein stabilizer
• 30% solution in water
• Made from IgG-free, protease-free Fraction V BSA
Bovine Serum Albumin Fraction V2201350 g• Commonly used blocking agent and antibody or protein stabilizer
• IgG-free, protease-free Fraction V BSA
Dry Milk Powder220124 x 25 g• Nonfat dry milk
• Commonly used blocking agent for western
10X Phosphate Buffered Saline (PBS)220204L Cubitainer®• Concentrated PBS Buffer
• RNase-, DNase-, protease-free (non-sterile)
Tween®-202200250 mL• Detergent commonly used for western blocking and washing
Mini Cell Scrapers22003Pack of 200• For harvesting cells or cell lysates from 96-, 48- and 24-well plates
• 0.5 cm (3/16″) wide and 6 cm (2 3/8″) long
• 20 packs of 10 scrapers per pack
• Polyethylene, disposable & sterile
Ultrafiltration Vials (3K MWCO)22018Pack of 5• For removing buffers, salts, and free dyes from proteins or DNA
• Simple microcentrifuge spin-column format
• 3 kDa molecular weight cut-off (MWCO)
Ultrafiltration Vials (10K MWCO)22004Pack of 5• For removing buffers, salts, and free dyes from proteins or DNA
• Simple microcentrifuge spin-column format
• 10 kDa molecular weight cut-off (MWCO)
DTT910501 g• Reducing agent commonly used to prepare samples for SDS-PAGE
TCEP910491 g• Odorless reducing agent
• More effective and stable than DTT
Cubitainer is a registered trademark of The Hedwin Division; Odyssey is a registered trademark of LI-COR, Inc; TWEEN is a registered trademark of Croda International PLC

 

Peacock™ and Peacock™ Plus Prestained Protein Markers on 10% Bis-Tris MES gels, labeled with apparent molecular weights of the bands.


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Buffers, Accessories, & Background Reducers

Buffers and other accessories for immunofluorescence staining and other applications.

Primary Antibodies

Growing collection of more than 1000 monoclonal antibodies. Many antibodies verified in HuProt™ Array and validated in IHC and other applications. Available with bright and photostable CF® dyes, R-PE, APC, PerCP, HRP, AP, biotin, or purified. Affordable 100 uL trial sizes.

Secondary Antibodies, Anti-Tag Antibodies, & Streptavidin Conjugates

Wide selection of secondary host and target species, anti-tag antibodies, and streptavidin conjugates. Choose from more than 20 CF® dyes, R-PE, APC, HRP, AP or biotin. Highly cross-adsorbed, F(ab’)2 fragments, and isotype-specific options. Affordable 50 uL trial sizes.