Shop All General Western Blot Reagents

TMB Substrate Solution Thermo Scientific™

Addition of sulfuric acid stop solution changes the color to yellow, enabling accurate measurement of the intensity at 450nm using a spectrophotometer or plate reader. This particular formulation is used in most of the Thermo Scientific Pierce ELISA Kits where it provides consistent performance for the sandwich ELISA technique.

Features of TMB Substrate Solution:

Product Number: N301
Size: 100 mL (sufficient for about ten 96-well microplates)
Storage: Shipped on cold packs. Store at 2-8°C immediately upon receipt. Do not use product beyond the stated expiration date.
Application: ELISA: Recommended for peroxidase-based enzyme immunoassays. Upon oxidation, TMB forms a water-soluble blue reaction product that can be measured spectrophotometrically at 605nm. Upon acidification, the reaction product becomes yellow with an absorbance peak at 450nm.
Precautions: This product is sensitive to contamination from a variety of oxidizing agents. To avoid contamination and premature expiration, avoid contacting TMB solution with any potential source of contamination. Never pipette directly from the bottle. Pour out required amount onto a tube and pipette from the tube. Do not return excess TMB to primary storage container.

Procedure:
• Add 100 µL of TMB substrate to each well containing peroxidase reaction.
• Incubate at room temperature for 15 to 30 minutes.
• Add 100 µL of stop solution (0.16M sulfuric acid, N600) to each well.
• Measure absorbance at 450nm.

Pierce™ 1-Step Transfer Buffer Thermo Scientific™

Thermo Scientific Pierce 1-Step Transfer Buffer is designed for rapid semi-dry transfer of 10-300kDa proteins from polyacrylamide gels (SDS-PAGE) to nitrocellulose or PVDF membranes using the Pierce G2 Fast Blotter.

Features of 1-Step Transfer Buffer:

Fast—high ionic strength formulation provides 5- to 10-minute protein transfer when used with compatible fast semi-dry blotting systems
Ready to use—supplied as a 1X solution that is stable and ready to use at room temperature
Optimized—designed for seamless and reliable performance with the Pierce G2 Fast Blotter
Compatible—effective with other semi-dry transfer devices equipped with a suitable high-current power supply, including the original Pierce Fast Semi-Dry Blotter (Part No. 88217)
Economical—excellent performance without the special or costly consumables that are required by other fast semi-dry transfer devices

When used with the Pierce G2 Fast Blotter, this buffer provides effective gel-to-membrane transfer of proteins in 5 to 10 minutes with efficiency that is equal to, or better than, conventional Western blotting techniques. Pierce 1-Step Transfer Buffer is also compatible with other protein semi-dry transfer devices, including the original Pierce Fast Semi-Dry Blotter (Part No. 88217), when they are paired with a suitable high-current power supply. Such devices provide constant high current (1.3 to 5.0 amps) to rapidly transfer proteins via the high ionic strength conditions supplied by the transfer buffer.

Requires:
Pierce G2 Fast Blotter (Part No. 62288) or other semi-dry transfer device that is equipped or paired with a capable high-current power supply.

Fast blotting methods require a high-current power supply, such as the Pierce G2 Fast Blotter, and an optimized high ionic strength transfer buffer, such as Pierce 1-Step Transfer Buffer. By increasing the current, excellent transfer efficiency can be achieved in much shorter time compared to conventional methods. Amperage is held at a constant rate based on the surface area of the transfer stack(s) (~22-23mA/cm2) and voltage is limited to 25V.

WesternBreeze™ Blocker/Diluent (Part A and B) Invitrogen™

The WesternBreeze® Blocker/Diluent (part A and B) is an optimized, easy-to-use blocker and primary antibody diluent system that yields low background/high signal western blot detection on nitrocellulose (NC) and polyvinylidene difluoride (PVDF) membranes. Sufficient reagents for 20 mini-blots.

WesternBreeze™ Chromogenic Kit, anti-mouse Invitrogen™

WesternBreeze® Chromogenic Kits detect proteins that have been immobilized on membranes (nitrocellulose or PVDF) following western transfer or bound directly from solution (dot blots). Detection is accomplished with a ready-to-use BCIP/NBT substrate for alkaline phosphatase. Non-fading purple precipitates develop at the protein bands on the membrane. WesternBreeze® chromogenic offers:

• Clear background
• High sensitivity-low picogram levels detectable
• High specificity
• One simple protocol-no optimization required

ZOOM™ Cathode Buffer 10X pH 9-12 Invitrogen™

The ZOOM® IEF Fractionator offers a fast, reliable method to reduce sample complexity, enrich low abundance proteins, and increase the dynamic range of detection. Solution phase isoelectric focusing with the ZOOM® IEF Fractionator provides reproducible separations in three hours. Fractionated samples are ready for further analysis by two dimensional gel electrophoresis (2DE), one dimensional gel electrophoresis (1DE), or two dimensional liquid chromatography/mass spectrometry (2D LC/MS).

The ZOOM® IEF Fractionator System (Figure 1) consists of the following components:

• ZOOM® IEF Fractionator is built for safety, reliability, and durability. It includes easy-to-assemble sample chambers, in-built buffer chambers, buffer trough, removable electrode assembly, and safety lid.
• ZOOM® Disks are pre-cast polyacrylamide gels that eliminate the need for manual preparation and minimize any chance of cross contamination. These immobilized buffered disks are pre-labeled, disposable, and designed for single use, ensuring consistent and reproducible fractionation. They resolve samples into as many as 7 fractions (using 8 disks of specific pH) to six fractions (using six disks of specific pH) from pH 3-12. Simply place each disk between the sample chambers in the ZOOM® IEF Fractionator to allow separation in a specific pH range.
• ZOOM® Reagents are proteomic grade reagents, including ZOOM® 2D Protein Solubilizers urea, thiourea, CHAPS, and ampholytes, that ensure optimal conditions for sample preparation.
• Basic Protein Kit which includes ZOOM® Discs and ZOOM Strips for IEF of basic proteins, focusing buffers, and solubilizer kits.

Sample Chamber and Fractionator Unit Set-up Procedure
Figures 4-6 illustrate assembly of the sample chambers and fractionator unit. The sample chamber and components fit together easily and offer a leak-proof seal. Simply place disks between the sample chambers in the ZOOM® IEF Fractionator to allow separation in a specific pH range. For example, if you want to fractionate proteins between pH 4.6 and 5.4, flank a sample chamber with a ZOOM (r) Disk of pH 4.6 and one of pH 5.4.

Downstream Processing by 2D Gel Electrophoresis
Following fractionation, the separated samples can be subjected to further analysis using narrow or broad range ZOOM® Strips on the ZOOM® IPGRunner™ System. Follow with second dimension analysis using neutral pH NuPAGE® ZOOM® Gels.

SuperSignal™ Western Blot Enhancer Thermo Scientific™

Thermo Scientific SuperSignal Western Blot Enhancer contains a membrane treatment reagent and a primary antibody diluent that increase both signal intensity and sensitivity 3- to 10-fold compared to a detection performed without it.

Features of SuperSignal Western Blot Enhancer:

Increase sensitivity—achieve 3- to 10-fold increase in signal intensity and sensitivity
Improve specificity—improves signal-to-noise ratio for poor quality and low affinity antibodies
Better clarity—reduces background for cleaner Western blots
Membrane compatibility—provides effective signal enhancement with PVDF and nitrocellulose membranes
Substrate compatibility—validated for use with chromogenic, chemiluminescent and fluorescent detection methods

When a protein or antigen is difficult to detect because of low abundance or poor immunoreactivity, use of SuperSignal Western Blot Enhancer can significantly reduce background and enhance detection of low-abundance and weakly immunoreactive antigens.

Novex™ pH 3-7 IEF Buffer Kit, Includes LC5300, LC5370, LC5371 Invitrogen™

Novex® pH 3-7 IEF Buffer Kit is optimized for using with Novex® pH 3-7 IEF gels. Each kit includes LC5300, LC5370, LC5371

Restore™ Western Blot Stripping Buffer Thermo Scientific™

Thermo Scientific Restore Western Blot Stripping Buffer safely and effectively removes primary and secondary antibodies from nitrocellulose and PVDF membranes to allow chemiluminescent Western blots to be reprobed.

Features of Restore Western Blot Stripping Buffer:

• Saves time—no need to re-run gels and blots
• Saves costly sample—re-probe the membrane using the same target sample
• Effective—formulation is more efficient at stripping antibodies than homemade buffers
• Gentle—does not damage the target antigen during stripping allowing efficient reprobing
• Odor-free—no mercaptans means no acrid odors
• Economical—less expensive than other commercial stripping buffers

Product Details
Performing gel electrophoresis and duplicate immunoblot assays to test new primary antibodies or antibody concentrations is time-consuming and expensive. Restore Western Blot Stripping Buffer eliminates this waste when detecting immunoblots with chemiluminescent Western blotting substrates. Restore Stripping Buffer provides clean and efficient removal of primary and secondary antibodies from immunoblots without removing or damaging the immobilized antigen allowing blots to be stripped and reprobed with confidence.

Chemiluminescent Western blot detection with reagents such as Thermo Scientific SuperSignal Substrates for horseradish peroxidase is one of the most common and sensitive methods in use today. Because these substrates do not precipitate and bind to membrane surfaces, Western blots detected by chemiluminescence can be stripped with reagents that remove affinity-bound primary and secondary antibodies. To be effective, a stripping buffer must be strong enough to disassociate bound antibodies but gentle enough to leave the transferred target proteins intact on the nitrocellulose or PVDF membrane. Restore Western Blot Stripping Buffer has these characteristics.

By stripping and reprobing, there is no need to waste rare or costly samples by running multiple gels in order to probe for different targets. A single membrane from one gel can be stripped with Restore Western Blot Stripping Buffer to remove the primary antibodies. Stripping the blot takes only 15 to 30 minutes, depending on the affinity of the primary antibody. After stripping, block and reprobe with a new primary antibody. Alternatively, a blot can be stripped and reprobed with adjusted antibody concentrations to optimize conditions after obtaining initially poor results.

Applications
• Reuse a nitrocellulose or PVDF blot to detect a different target with a different primary antibody
• Reprobe a blot to correct or optimize antibody concentrations that were ineffective the first time

Protocol Summary
• Wash blot to remove chemiluminescent substrate.
• Incubate blot in Restore Western Blot Stripping Buffer for 5 to 15 minutes at 37°C (room temperature is sufficient for some antibodies).
• Remove blot and wash in Wash Buffer (TBS-T or PBS-T).
• Test for sufficient removal of antibodies.
• Perform next immunoblot experiment.

No-Stain™ Protein Labeling Reagent Invitrogen™

The Invitrogen No-Stain Protein Labeling Reagent provides a flexible, accurate, rapid, and reliable method to visualize and normalize proteins in a gel or on a membrane (post-transfer). It forms covalent bonds to proteins in gels or on membranes within 10 minutes, does not require any de-staining steps, and can be instantly visualized using any commonly available imager. No-Stain reagent does not require any particular gels or other reagents and is compatible with gel stains and western workflows.

Instant visualization of proteins in gels
Coomassie and other gel staining and de-staining steps can be extremely time consuming and cumbersome. No-Stain Protein Labeling Reagent forms covalent bonds with the lysine amino acid side chains on all proteins in a gel within 10 minutes. Since lysine is one of the most abundant amino acids, No-Stain reagent enables detection of all proteins in a gel or on a membrane, and the strong signal emission from the covalently bonded reagent provides nanogram-level sensitivity.

Alternative to traditional gel staining reagents—provides more accurate normalization over a wide range of protein lysate concentrations (1–80 µg)
Sensitive—lower limit of detection of 20 ng per band
Specific—forms bonds only with the lysine side chains of proteins. Unbound reagent does not emit, thereby enabling a superior signal-to-noise ratio
Flexible—no need to change your gels to get stain-free convenience. No-Stain reagent provides stain-free convenience with any gel type (precast or pour-your-own gel)

Achieve the gold standard for quantitative western blotting
Protein normalization is a critical step in obtaining reliable and reproducible quantitative western blotting. Total protein normalization is considered the gold standard for quantitative western blotting. Many leading journals have developed guidelines for submitting western blotting research and select quotes from those guidelines are provided below.

• “For quantitative comparisons, appropriate reagents, controls and imaging methods with linear signal ranges should be used” – Nature
• “Record how data were obtained, whether signal intensity was linear with antigen loading, and how protein loading was normalized” – Journal of Biological Chemistry
• “Normalize signal intensity to total protein loading (assessed by staining membranes for total protein) whenever possible” – Journal of Biological Chemistry
• “House-keeping proteins should not be used for normalization without evidence that manipulations do not affect expression” – Journal of Biological Chemistry

An accurate loading control should display a linear relationship between signal intensity and sample load in all experimental conditions. The signal intensity obtained from labeling of total proteins on a membrane with No-Stain reagent ensures a linear relationship between signal intensity and sample load (see figure below) in all experimental conditions. Therefore, the use of No-Stain reagent in quantitative western blot applications enables the use of total protein as an ideal loading control.

Easy-to-use protocol—labeling of proteins within 10 minutes on either nitrocellulose or PVDF membranes
Rapid visualization using a wide-range of imagers with UV or fluorescence light sources
Accurate total protein normalization—the broad linear range for protein detection of 1–80 μg enables detection of No-Stain signal along with that of your target protein to achieve accurate total protein normalization
Sensitive and stable—nanogram level sensitivity with a stable signal that is compatible with downstream immunodetection steps
Superior analysis—housekeeping proteins are susceptible to signal saturation and other biological variations which are not observed when using No-Stain reagent for total protein normalization

Learn more about No-Stain Protein Labeling Reagent ›

Restore™ PLUS Western Blot Stripping Buffer Thermo Scientific™

Thermo Scientific Restore PLUS Western Blot Stripping Buffer is an advanced formula for removing bound primary and secondary antibodies from membranes so they can be reprobed and detected with chemiluminescent substrates.

Features of Restore PLUS Western Blot Stripping Buffer:

Ready and easy to use—no dilution necessary; no offensive odors; store at room temperature
Compatible—use on nitrocellulose and PVDF membranes, whether still wet or already dry; works with practically any blocking buffer, enzyme conjugate and chemiluminescent substrate
Cost effective—save valuable time and samples; strip blots effectively the first time
Robust yet gentle—transferred proteins remain viable; strip the same blot up to five times
Flexible—strip and reprobe to optimize antibody concentrations or to detect a new antigen with different antibodies

Restore PLUS Buffer is an alternative formulation of the original Thermo Scientific Restore Western Blot Stripping Buffer. Restore PLUS Stripping Buffer was designed for use with antibodies that are difficult to remove from western blots and require longer incubation times or incubation temperatures greater than 22°C with gentler formulations. High-affinity antibodies can be quickly and effectively stripped from western blots at room temperature without removing transferred proteins, thereby allowing multiple reprobes of the target.

Protocol Summary:
• Wash blot to remove chemiluminescent substrate
• Incubate blot in Restore Western Blot Stripping Buffer for 5 to 15 minutes at room temperature
(or incubate at 37°C for high affinity antibodies)
• Remove blot and wash in Wash Buffer
• Block membrane
• Test for sufficient removal of antibodies
• Perform next immunoblot experiment

Pierce™ Western Blot Signal Enhancer Thermo Scientific™

Thermo Scientific Pierce Western Blot Signal Enhancer is a two-reagent system for conditioning protein blots after transfer to greatly enhance the effectiveness of primary antibodies and intensify the final detection signal in Western blot experiments.

Features of Western Blot Signal Enhancer:

Increases protein detection—most protein targets show a three- to 10-fold increase in signal intensity, enabling much less protein to be detected with the same substrate and method
Improves antibody binding—the membrane-treatment reagent exposes and conditions target proteins so that specific antibodies can bind more effectively
Works for nearly any protein—signal enhancement has been demonstrated with targets such as IL-6, p53, NFκB, BRCA1 and EGF
Effective with any substrate—enhances both chemiluminescent (ECL) and colorimetric detection for Western blots
Compatible with any membrane—enhances signal on nitrocellulose and PVDF membrane, regardless of pore size (enhancement is less pronounced with PVDF)
Fast, 15-minute protocol—optimized for a combination of simplicity, speed and signal enhancement for most proteins
Ready-to-use—no formulating or diluting necessary, and the reagents are stable for storage at room temperature

The Pierce Western Blot Signal Enhancer membrane treatment procedure is very simple, takes only 15 minutes and can be added to nearly any existing Western blotting protocol. The result is an increase in the intensity of target protein bands on the Western blot or detection of target proteins at levels that were previously not possible. The product is effective for signal intensification with both chemiluminescent and chromogenic substrates, especially with nitrocellulose membranes.

WesternBreeze™ Chromogenic Kit, anti-goat Invitrogen™

WesternBreeze® Chromogenic Kits detect proteins that have been immobilized on membranes (nitrocellulose or PVDF) following western transfer or bound directly from solution (dot blots). Detection is accomplished with a ready-to-use BCIP/NBT substrate for alkaline phosphatase. Non-fading purple precipitates develop at the protein bands on the membrane. WesternBreeze® chromogenic offers:

• Clear background
• High sensitivity-low picogram levels detectable
• High specificity
• One simple protocol-no optimization required

Novex™ Reversible Membrane Protein Stain Kit Invitrogen™

Novex® Reversible Protein Stain is a highly sensitive stain used for determining protein transfer efficiency after blotting. Quantitative and completely reversible, this stain is compatible for use with both nitrocellulose (NC) and polyvinylidene difluoride (PVDF) membranes.

Incubation Tray, 10 x 14 cm Invitrogen™

The Incubation Tray enables convenient and even staining of E-PAGE™ blots. The tray is 10 cm (l) x 14 cm (w) x 3 cm (d) with a lid. It is suitable for use with immunodetection or blot stains.

Restore™ Western Blot Stripping Buffer, Trial Size Thermo Scientific™

Thermo Scientific Restore Western Blot Stripping Buffer safely and effectively removes primary and secondary antibodies from nitrocellulose and PVDF membranes to allow chemiluminescent Western blots to be reprobed.

Features of Restore Western Blot Stripping Buffer:

• Saves time—no need to re-run gels and blots
• Saves costly sample—re-probe the membrane using the same target sample
• Effective—formulation is more efficient at stripping antibodies than homemade buffers
• Gentle—does not damage the target antigen during stripping allowing efficient reprobing
• Odor-free—no mercaptans means no acrid odors
• Economical—less expensive than other commercial stripping buffers

Product Details
Performing gel electrophoresis and duplicate immunoblot assays to test new primary antibodies or antibody concentrations is time-consuming and expensive. Restore Western Blot Stripping Buffer eliminates this waste when detecting immunoblots with chemiluminescent Western blotting substrates. Restore Stripping Buffer provides clean and efficient removal of primary and secondary antibodies from immunoblots without removing or damaging the immobilized antigen allowing blots to be stripped and reprobed with confidence.

Chemiluminescent Western blot detection with reagents such as Thermo Scientific SuperSignal Substrates for horseradish peroxidase is one of the most common and sensitive methods in use today. Because these substrates do not precipitate and bind to membrane surfaces, Western blots detected by chemiluminescence can be stripped with reagents that remove affinity-bound primary and secondary antibodies. To be effective, a stripping buffer must be strong enough to disassociate bound antibodies but gentle enough to leave the transferred target proteins intact on the nitrocellulose or PVDF membrane. Restore Western Blot Stripping Buffer has these characteristics.

By stripping and reprobing, there is no need to waste rare or costly samples by running multiple gels in order to probe for different targets. A single membrane from one gel can be stripped with Restore Western Blot Stripping Buffer to remove the primary antibodies. Stripping the blot takes only 15 to 30 minutes, depending on the affinity of the primary antibody. After stripping, block and reprobe with a new primary antibody. Alternatively, a blot can be stripped and reprobed with adjusted antibody concentrations to optimize conditions after obtaining initially poor results.

Applications
• Reuse a nitrocellulose or PVDF blot to detect a different target with a different primary antibody
• Reprobe a blot to correct or optimize antibody concentrations that were ineffective the first time

Protocol Summary
• Wash blot to remove chemiluminescent substrate.
• Incubate blot in Restore Western Blot Stripping Buffer for 5 to 15 minutes at 37°C (room temperature is sufficient for some antibodies).
• Remove blot and wash in Wash Buffer (TBS-T or PBS-T).
• Test for sufficient removal of antibodies.
• Perform next immunoblot experiment.
Results per page
    spinner