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WESTERN BLOTTING

Reagents, buffers and accessories

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
Cell permeable nuclear dye for In-Cell Western™ normalization
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 chemiluminescence 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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VersaBlot™ Near-IR Total Protein Normalization Kits

VersaBlot™ Total Protein Normalization 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. Proteins can then be visualized on a gel or membrane using a fluorescent gel scanner, allowing detection of as little as 1 ng protein per band. If desired, the prestain can be reversed after scanning, facilitating downstream multi-color western blot analysis. 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.

VersaBlot™ Total Protein Prestain Features

  • Reversible prestain for downstream multi-color western blot analysis
  • 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 and 10% difference in protein content

In-gel fluorescence image of bovine serum albumin (BSA) labeled with the VersaBlot™ total protein normalization 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.
VersaBlot™ total protein normalization 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 VersaBlot™ total protein normalization kits show better linearity compared to housekeeping protein detection.

Product Information

ProductCatalog numberSizeAbs/EmImaging Systems / Detection channels
VersaBlot™ CF®680T Total Protein Normalization Kit
33025-T100 labelings681 / 698 nmAmersham Typhoon™ Trio Cy®5 channel
Amersham Typhoon™ 5 IR short channel
LI-COR® Odyssey® 700 channel
33025500 labelings
VersaBlot™ CF®770T Total Protein Normalization Kit33026-T100 labelings764 / 787 nmAmersham Typhoon™ 5 IR long channel
LI-COR® Odyssey® 800 channel
33026500 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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RedDot™1 Far-Red Nuclear Stain for In-Cell Western™

RedDot™1 is a far-red cell membrane-permeable nuclear dye similar to Draq5™. Staining does not require a wash step and demonstrates greater photostability than the traditional blue fluorescent nuclear stains DAPI and Hoechst. RedDot™1 can be excited at a wide range of wavelengths between 488 nm and 647 nm, with an emission maximum at 694 nm. RedDot™1 is particularly useful for cell number normalization for In-Cell Western™ assays using a near-infrared scanner such as the LI-COR® Odyssey®. In fixed cells, RedDot™ 1 staining generates a highly linear fluorescence signal in the 700 nm channel that is proportional to the number of cells. In a direct comparison with DRAQ5™/Sapphire700™,  RedDot™ 1 staining demonstrates a higher signal and a wider linear range.

RedDot™1 Features

  • Ideal for normalization of In Cell Western® in fixed cells, similar to Draq5™/Sapphire®700
  • Highly thermostable and photostable, for convenient handling and demanding imaging applications
  • Can also be used for DNA content analysis by flow cytometry like Vybrant™ DyeCycle™ Ruby
  • λExEm = 662/694 nm (with DNA), for detection in the Cy®5 channel

RedDot1 far-red nuclear stain, 200X in H2O

RedDot1 far-red nuclear stain, 200X in H2O

RedDot™1 staining of HeLa cells for cell number normalization. HeLa cells were seeded in 96 wells at the indicated densities. After 24 hours, cells were fixed, permeabilized, and stained with the indicated dyes for one hour at room temperature according to the supplier’s protocol for DRAQ5™/Sapphire700™. Linearity of fluorescence signal for 0-6250 cells per well (left). Linearity of fluorescence signal for 0-50,000 cells per well (right). HeLa cells seeded at 25,000 cells per well were confluent at the time of assay.

ProductCatalog No.SizeColor (Ex/Em)Applications
RedDot™1 Far-Red Nuclear Stain, 200X in Water40060-T25 uL(Far-red)
662/694 nm
• Live cell nuclear stain comparable to Draq5™

• Ideal for In-Cell Western® normalization

• Suitable for microscopy and cell cycle analysis by flow cytometry
40060250 uL
40060-11 mL
Draq5 is a trademark of BioStatus Ltd. In Cell Western and Sapphire are registered trademarks of LI-COR Inc.

 

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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
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.

Western Blotting Reagents

TrueBlack® WB Blocking Buffer offer superior signal-to-noise for fluorescent and near-infrared western blots. VersaBlot™ Total Protein Normalization Kits allow highly linear normalization for near-infrared WB.

Protein Gel Stains

One-Step Blue™ & One-Step Lumitein™ protein gel stains are simple, rapid, non-toxic, and low-cost visible and fluorescent gel stains.

Protein Markers

Peacock™ protein markers have multi-color bands ranging from 8 to 245 kDa. Peacock™ Plus available with a wider molecular weight range.