Western Blotting

Reagents, buffers and accessories

Explore Western Blotting Solutions

Near-IR CF® Dyes for WB

Antibody conjugates of our best-in-class NIR dyes

TrueBlack® WB Blocking Buffer

For superior signal-to-noise in fluorescent western

Total Protein Prestain

Super-sensitive & linear near-IR protein quantitation in gels or blots

Far-Red Nuclear Stain

Nuclear dye that offers superior linearity for In-Cell Western® normalization

Chromogenic Detection

Substrates plus secondary & anti-tag antibodies for HRP & alkaline phosphatase

Buffers & Accessories

Prestained protein markers, buffers, & detergents

Near-IR CF® Dye conjugates for Western Blotting

Near-infrared (near-IR) western detection is highly sensitive and offers advantages of a wider linear range and multiplexing capability compared to chemiluminescence detection. 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 a 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 tra... See More

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 an... See More

Near-IR CF® Dye Secondary Antibodies for Multiplex WB

Conjugate	Cross-adsorption	CF®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-Goat	Ck, GP, Hs, Hu, Ms, Rb, Rt, SHm	20060	20196	20362	20277
Donkey Anti-Guinea Pig	Bv, Ck, Gt, Hs, Hu, Ms, Rb, Rt, Shp, SHm	20241			20242
Donkey Anti-Mouse	Bv, Ck, Gt, GP, Hs, Hu, Rb, Shp, SHm		20194			20363
Donkey Anti-Rabbit	Bv, Ck, Gt, GP, Hs, Hu, Ms, Rt, Shp, SHm	20418	20195	20298	20484	20344
Donkey Anti-Sheep	Ck, GP, Hs, Hu, Ms, Rb, Rt, SHm	20062
Goat Anti-Guinea Pig	Bv, Ck, Gt, Hs, Hu, Ms, Rb, Rt, SHm, Shp	20497	20496	20499	20498
Goat Anti-Mouse	Bv, Hs, Hu, Rb, Sw	20065	20192	20463	20077	20342
Goat Anti-Mouse IgG1	Bv, Hu, Rb	20253			20254
Goat Anti-Mouse IgG2a	Bv, Hu, Rb	20263	20842		20264
Goat Anti-Mouse IgG2b	Bv, Hu, Rb	20273		20430	20274
Goat Anti-Rabbit	Hu, Ms, Rt	20067	20193		20078	20343
Goat Anti-Rat	Bv, Hs, Hu, Rb	20069			20383
Rabbit Anti-Mouse	Hu	20061

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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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 (Figure 1)
Reduces non-specific protein bands and background over the entire membrane
Suppresses background from charged dyes better than BSA, gelatin, or casein
PVDF- and nitrocellulose membranes-compatible
Free of mammalian proteins
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®... See More

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 ... See More

Superior Western Blocking for Fluorescent Dyes

Non-specific signal in WB can arise from multiple sources, including antibody cross-reactivity, non-specific antibody adsorption, and membrane autofluorescence. Another potential cause of background is the fluorescent dyes themselves on the specificity of labeled antibodies. Highly charged dyes have improved solubility and brightness of conjugates compared to uncharged dyes. However, the extra charge carried by labeled antibodies can result in non-specific binding to proteins and membranes. TrueBlack® WB Blocking Buffer Kits block background from multiple sources, including charged dye conjugates (Figure 2). The TrueBlack® WB 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.

TrueBlack® WB Blocking Buffer Kit vs. Odyssey® Blocking Buffer

Product	TrueBlack® WB Blocking Buffer Kit	Odyssey® Blocking Buffer
Trial Size	For 10 membranes	125 mL for 4 membranes
Full Size	For 50 membranes	500 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.

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TrueBlack® WB Blocking Buffer Kit

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

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 label purified proteins or cell lysates with near-infrared CF® Dyes before running samples on SDS-PAGE. Proteins can be visualized on a gel or membrane using a fluorescent gel scanner. The prestain can be reversed, facilitating downstream multi-color western blot analysis.

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 la... See More

VersaBlot™ Total Protein Normalization Kits

Product	Catalog number	Size	Abs/Em	Imaging Systems / Detection channels
VersaBlot™ CF®680T Total Protein Normalization Kit	33025-T	100 labelings	681 / 698 nm	Amersham Typhoon™ Trio Cy®5 channel Amersham Typhoon™ 5 IR short channel LI-COR® Odyssey® 700 channel
VersaBlot™ CF®680T Total Protein Normalization Kit	33025	500 labelings	681 / 698 nm
VersaBlot™ CF®770T Total Protein Normalization Kit	33026-T	100 labelings	764 / 787 nm	Amersham Typhoon™ 5 IR long channel LI-COR® Odyssey® 800 channel
VersaBlot™ CF®770T Total Protein Normalization Kit	33026	500 labelings	764 / 787 nm

Typhoon is a trademark and Cy dye is a registered trademark of Cytiva; 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-permeant nuclear dye similar to Draq5™. Compared to DRAQ5™/Sapphire700™, it delivers higher signal intensity, a broader linear range, and does not require a wash step. RedDot™1 is useful for normalizing cell numbers in In-Cell Western® assays using a near-infrared scanner. In fixed cells, RedDot™ 1 staining generates a highly linear fluorescence signal that is proportional to the number of cells.

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
λEx/λEm = 662/694 nm (with DNA), for detection in the Cy®5 channel

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.

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RedDot™1 Far-Red Nuclear Stain

Chromogenic Detection

Chromogenic detection by horseradish peroxidase (HRP) or alkaline phosphatase is a widely used and cost-effective method for Western blot analysis. Colorimetric detection works by the production of an insoluble colored byproduct from a chemical reaction between a substrate and reporter enzyme. Biotium offers several secondary and anti-tag antibodies conjugated to either HRP or alkaline phosphatase for chromogenic detection. See the tables below for available conjugates. You can also view our entire Secondary Antibody Product Listings for other HRP and alkaline phosphatase conjugates, including anti-human IgG, IgA, and IgM antibodies. Biotium also offers several colorimetric substrates for alkaline phosphatase and a DAB substrate kit for HRP, listed in the table below.

HRP and Alkaline Phosphatase Secondary Antibodies

Conjugate	Donkey Anti-Mouse (Min X Rat)	Donkey Anti-Rabbit	Goat Anti-Chicken	Goat Anti-Mouse	Goat Anti-Rabbit	Goat Anti-Rat	Chicken Anti-Goat	Goat Anti-Llama	Goat Anti-Mouse	Goat Anti-Rabbit
Cross-Adsorption	Bv, Ck, Gt, GP, Hs, Hu, Rb, Rt, Shp, SHm	Bv, Ck, Gt, GP, Hs, Hu, Ms, Rt, Shp, SHm	Bv, Gt, GP, Hs, Hu, Ms, Rb, Rt, SHm, Shp	Bv, Hs, Hu, Rb, Sw	Hu, Ms, Rt	Bv, Hs, Hu, Rb	None	None	None	None
HRP	20404	20405	20474	20401	20403	20406	20839	20475	20400	20402
Alkaline Phosphatase	20466	20467							20464	20465

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HRP and Alkaline Phosphatase Anti-Tag Antibodies

Conjugate	Streptavidin Conjugates	Mouse Anti-Biotin (Hyb8)	Mouse Anti-Biotin (BTN/2032R)	Mouse Anti-Myc Tag (9E10.3)	Rabbit Anti-Myc Tag (MYC2895R)	Goat Anti-DIG	Rabbit Anti-DNP
HRP	29049	BNCH0400	BNCH2032	BNCH0596	BNCH2865	20864	20871
Alkaline Phosphatase	29071	BNCAP0400	BNCAP2032	BNCAP0596	BNCAP2895

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.

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Chromogenic Substrates for HRP and Alkaline Phosphatase

Product	Catalog No.	Size	Substrate for	Precipitate Color
BCIP, NA	1001	100 mg	Alkaline Phosphatase	Dark Blue
	1001-1	500 mg
	1001-2	5 g
BCIP, Toluidine	10002	100 mg	Alkaline Phosphatase	Dark Blue
	10002-1	500 mg
	10002-2	5 g
BCIP Pink	10006	100 mg	Alkaline Phosphatase	Pink
BCIP Pink/NBT Kit	10007	1 set	Alkaline Phosphatase	Pink
BCIP Red	10004	100 mg	Alkaline Phosphatase	Red
BCIP Red/NBT Kit	10005	1 set	Alkaline Phosphatase	Red
BCIP/NBT Kit	10003	1 set	Alkaline Phosphatase	Dark Blue
Chromogenic Phosphatase Substrate Sampler	10022	1 kit	Alkaline Phosphatase	Multiple
DAB Substrate Kit	30015	1 kit	HRP	Brown

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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	Catalog No.	Size	Features
Peacock™ Prestained Protein Marker	21530	50 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 Marker	21531	50 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
4X Protein Loading Buffer with Orange Tracking Dye	40136	15 mL	• A convenient 4X sample loading buffer for protein gel electrophoresis • Optimized for use with denaturing SDS-PAGE • Orange tracking dye avoids unwanted background fluorescence caused by blue tracking dyes
Ponceau S Solution	22001	1 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 Kit	23013	For 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 Agent	22010	100 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 Powder	22011	2 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% Solution	22014	100 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 V	22013	50 g	• Commonly used blocking agent and antibody or protein stabilizer • IgG-free, protease-free Fraction V BSA
Dry Milk Powder	22012	4 x 25 g	• Nonfat dry milk • Commonly used blocking agent for western
Tween®-20	22002	50 mL	• Detergent commonly used for western blocking and washing
Mini Cell Scrapers	22003	Pack 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)	22018	Pack 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)	22004	Pack 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)
DTT	91050	1 g	• Reducing agent commonly used to prepare samples for SDS-PAGE
TCEP	91049	1 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

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Product Spotlight

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Tool

CF® Dye Selection Tool

Find the optimal CF® Dye for a specific laser/filter combination for flow cytometry.

Blog

Your Go-To Guide for Protein Stains

Deciding between Coomassie or silver stain for that gel? Check out this helpful guide.

Protocol

Protocol: Fluorescent Western Blotting

A basic protocol for fluorescent western detection.

Tech Tip

Tech Tip: Troubleshooting Tips for Fluorescence Staining

Solutions for common microscopy, flow cytometry, or western problems.

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FAQs

Is protein staining on SDS-PAGE gels with the One-Step protein stains compatible with western blotting?

We do not recommend staining proteins on the polyacrylamide gel with the One-Step protein gel stains before western blotting as it can significantly reduce protein transfer to the membrane. If protein detection on the gel prior to transfer is desired, the Total Protein Prestains would be more suitable.

The 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. After electrophoresis, the bands can be visualized on the gel 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 and staining is reversible.

My product was accidentally left out at room temperature or exposed to light. Is it ruined?

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, 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.

One exception that we are aware of is GelGreen^®, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen^™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.

What do highly cross-adsorbed and “min x” mean?

“Min x” followed by a list of species means that the antibody has minimal cross-reactivity against antibodies from those species. The antibody has been adsorbed against serum proteins from the listed species, to ensure that it does not cross-react with those species.

Highly cross-adsorbed antibodies are recommended when doing multiple staining with antibodies from different species (for example, co-staining with two different primary antibodies from mouse and rat hosts) or when staining tissues from species that are closely related to the antibody host (such as using mouse antibodies to stain rat tissue).

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