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LightShift™ EMSA Optimization and Control Kit (Thermo Scientific™)

The Thermo Scientific LightShift EMSA Optimization and Control Kit is an extraordinarily robust and sensitive system for performing electrophoretic mobility shift assays (EMSA) to identify and characterize protein-DNA binding interactions. The kit includes reagents for setting up and customizing DNA binding reactions and a control set of DNA and protein extract to test the kit system.

Features of theLightShift EMSA Optimization and Control Kit:

• Excellent for detecting low-abundance proteins in nuclear extracts
• Sensitivity that surpasses radioactive and digoxigenin methods
• Compatible with previously established binding conditions for popular DNA-protein interactions
• Includes EBNA control system to help new users develop a working assay and understand the methods used to confirm binding interaction specificity

The principle for LightShift EMSA Detection is similar to a Western blot. Biotin end-labeled duplex DNA is incubated with a nuclear extract or purified factor and electrophoresed on a native gel. The DNA is then rapidly (30 minutes) transferred to a positive nylon membrane, UV crosslinked, probed with streptavidin-HRP conjugate and incubated with the substrate. The protocol from labeling to results can be accomplished in a single day.

The interaction of proteins with DNA is central to the control of many cellular processes including DNA replication, recombination and repair, transcription, and viral assembly. One technique that is central to studying gene regulation and determining protein:DNA interactions is the electrophoretic mobility shift assay (EMSA).

The EMSA technique is based on the observation that protein:DNA complexes migrate more slowly than free DNA molecules when subjected to non-denaturing polyacrylamide or agarose gel electrophoresis. Because the rate of DNA migration is shifted or retarded upon protein binding, the assay is also referred to as a gel shift or gel retardation assay. Until conception of the EMSA protein:DNA interactions were studied primarily by nitrocellulose filter-binding assays.

All that is needed to perform the assay is purified DNA target that has been end-labeled with biotin, the protein extract to be tested, nylon membrane and basic electrophoresis equipment. DNA targets can be synthesized with 5' or 3' biotin labels or they can be labeled after synthesis using the Thermo Scientific Biotin 3' End DNA Labeling Kit (Product No. 89818). Nuclear, cytosolic or whole cell protein extracts can be obtained by a variety of methods, including the Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Product No. 78833).

More Product Data
Transfer EMSA gels using the Pierce G2 Fast Blotter

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LightShift™ Chemiluminescent EMSA Kit
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Rapid ELISA Mouse mAb Isotyping Kit (Thermo Scientific™)

The Thermo Scientific Pierce Rapid ELISA Mouse mAb Isotyping Kit provides antigen-independent determination of mouse monoclonal antibody isotype using a microplate method that enables multiple samples to be tested in about one hour.

Features of the Rapid ELISA Mouse mAb Isotyping Kit:

Fast—determine antibody isotype in approx. 1 hour; considerably faster than traditional ELISA kits
Convenient—eight-well strip format allows partial use of the plate; use one strip (column) for each sample (12 samples per plate)
Specific—characterize antibodies for six different subclasses and two different light-chain types
Sensitive—accurate characterization with samples containing at least 3 ng/mL of test antibody
Flexible—kit is compatible with hybridoma cell culture supernatant, ascites fluid or purified antibodies
Cost effective—cost per assay is less than with single-use assay strips or cassettes
No special equipment needed—assess results visually or measure quantitatively using an ordinary ELISA plate reader (450nm)
Complete and easy to use—includes precoated plates, detecting antibody, buffers, and TMB substrate and stop solutions

This fast assay uses ELISA strip-plates that are pre-coated in different wells with anti-mouse heavy-chain capture antibodies (anti-IgG1, IgG2a, IgG2b, IgG3, IgA and IgM) or anti-mouse light-chain capture antibody (kappa or lambda). This approach eliminates the need to purify and immobilize an antigen to determine immunoglobulin isotype (antibody subclass and light chain identity). A mouse monoclonal antibody sample applied to the wells can be isotyped within one hour. Results are evaluated qualitatively by visual inspection or quantitatively by measuring the absorbance at 450nm.

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Pierce™ Bovine Serum Albumin Standard, 2 mg/mL (Thermo Scientific™)

Thermo Scientific Pierce BSA Protein Assay Standards are high-quality reference samples for generating accurate standard curves and calibration controls in total protein assays.

Features of BSA Protein Assay Standards:

Convenient—50 mL bottle
Universal—recognized as the industry standard for protein quantitation in colorimetric protein assays
Pure and stable—supplied in ultrapure 0.9% saline solution with 0.05% sodium azide; room temperature stable
Accurate and consistent—precisely formulated at 2.00 +/-0.03 mg/mL compared to an NIST reference

These bovine serum albumin (BSA) solutions are protein concentration reference standards for use in BCA, Bradford and other protein assay protocols. BSA is the universally accepted reference protein for total protein quantitation. The albumin standard is precisely formulated at 2 mg/mL in an ultrapure 0.9% sodium chloride (saline) solution. The concentration of the stock solution is calibrated by direct comparison to purified BSA (Fraction V) from the National Institute of Standards and Technology (NIST).

Applications:
• Protein assay quantitation standard (BCA Protein Assay, Coomassie-Bradford Assay, etc.)
• Protein recovery control for desalting and other column procedures
• General calibration of spectrophotometer UV-lamp (absorbance at 280nm)

Selection of a protein standard is potentially the greatest source of error in any protein assay. The best choice for a standard is a purified, known concentration of the most abundant protein contained in the samples being tested. Often, a highly purified, known concentration of the protein of interest is not available or it is too expensive to use as the standard, or the sample itself is a mixture of many proteins (e.g., cell lysate). In such cases, the best standard is one that will produce a normal (i.e., average) color response curve with the selected protein assay method and is readily available to any researcher. BSA is such a protein, and the Pierce Albumin Standards are the most convenient source of ready-to-use BSA standard.

For greatest accuracy in estimating total protein concentration in unknown samples, it is essential to include a standard curve each time the assay is performed. This is particularly true for the protein assay methods that produce non-linear standard curves. Deciding on the number of standards and replicates used to define the standard curve depends upon the degree of non-linearity in the standard curve and the degree of accuracy required. In general, fewer points are needed to construct a standard curve if the color response is linear. Typically, standard curves are constructed using at least two replicates for each point on the curve.

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Pierce™ Bovine Serum Albumin Standard Pre-Diluted Set

Pierce™ Coomassie (Bradford) Protein Assay Kit (Thermo Scientific™)

The Thermo Scientific Pierce Coomassie Protein Assay Kit is a ready-to-use, stable formulation of the traditional Bradford assay reagent to measure (A595 nm) total protein concentration compared to a protein standard.

The Pierce Coomassie Protein Assay Kit is a ready-to-use formulation of the popular assay reagent originally described by Bradford in 1976. When mixed with a protein solution, the acidic coomassie-dye reagent changes color from brown to blue in proportion the amount of protein present in the sample. Protein determinations are made by comparison to the color response of protein assay standards, usually prepared as a series of known dilutions of bovine serum albumin (BSA) or bovine gamma globulin (BGG). The kit includes Coomassie Protein Assay Reagent and a package of Albumin Standard Ampules. The simple procedure is adaptable to nearly any volume scale, including test tubes, cuvettes and microplates.

Features of the Coomassie Protein Assay Kit:

Bradford reagent—stable, ready-to-use kit of the classical Bradford assay reagent
Colorimetric—measure with a standard spectrophotometer or plate reader (595 nm)
Easy to use—single reagent; no working reagent preparation required
Fast—almost immediate color development; add, mix and read results
Broad range—detects protein concentration in the range 1 to 1500 µg/mL
Flexible—microplate and cuvette protocols provided with the instructions and adaptable to several target working ranges

How the Coomassie (Bradford) Assay Detects Protein
Use of coomassie G-250 dye in a colorimetric reagent for the detection and quantitation of total protein was first described by Dr. Marion Bradford in 1976. In the acidic environment of the reagent, protein binds to the coomassie dye. This results in a spectral shift from the reddish/brown form of the dye (absorbance maximum at 465nm) to the blue form of the dye (absorbance maximum at 610 nm). The difference between the two forms of the dye is greatest at 595 nm, so that is the optimal wavelength to measure the blue color from the coomassie dye-protein complex. If desired, the blue color can be measured at any wavelength between 575 nm and 615 nm. At the two extremes (575 nm and 615 nm) there is a loss of about 10% in the measured amount of color (absorbance) compared to that obtained at 595 nm.

Development of color in coomassie dye-based (Bradford) protein assays has been associated with the presence of certain basic amino acids (primarily arginine, lysine and histidine) in the protein. Van der Waals forces and hydrophobic interactions also participate in the binding of the dye by protein. The number of Coomassie dye ligands bound to each protein molecule is approximately proportional to the number of positive charges found on the protein. Free amino acids, peptides and low molecular weight proteins do not produce color with coomassie dye reagents. In general, the mass of a peptide or protein must be at least 3000 daltons to be assayed with this reagent. The assay is performed at room temperature and no special equipment is required. Simply add the sample to the tube containing reagent and the resultant blue color is measured at 595 nm following a short room-temperature incubation. The coomassie dye containing protein assay is compatible with most salts, solvents, buffers, thiols, reducing substances and metal chelating agents encountered in protein samples.

For more information, see the article "Chemistry of Protein Assays" in the Protein Methods Library.

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Ampule Breakers

Pierce™ Bovine Gamma Globulin Standard Ampules, 2 mg/mL (Thermo Scientific™)

Thermo Scientific Pierce BGG Protein Assay Standards are high-quality reference samples for generating accurate standard curves and calibration controls in total protein assays.

Features of BGG Protein Assay Standards:

Convenient—1 mL ampules
Antibody standard—best reference standard for immunoglobulin quantitation in colorimetric protein assays
Bradford standard—best general protein standard in coomassie-based (Bradford) protein assays
Pure and stable—supplied in ultrapure 0.9% saline solution with 0.05% sodium azide; room temperature stable
Accurate and consistent—precisely formulated at 2.00 +/-0.03 mg/mL, ensuring excellent lot-to-lot consistency

These bovine gamma globulin (BGG) solutions are protein concentration reference standards for use in BCA, Bradford and other protein assay protocols. BGG is an accepted reference protein for total protein quantitation of purified antibodies or immunoglobulin-rich samples. The IgG standard is precisely formulated at 2 mg/mL in an ultrapure 0.9% sodium chloride (saline) solution. The concentration of the stock solution of purified BGG (Fraction II) is calibrated by direct comparison to an internal standard to ensure lot-to-lot consistency and accuracy.

Applications:
• Protein assay immunoglobulin quantitation standard (Coomassie-Bradford Assay, etc.)
• Antibody recovery control for desalting and other column procedures
• Loading control for SDS-PAGE
• General calibration of spectrophotometer UV-lamp (absorbance at 280nm)

Selection of a protein standard is potentially the greatest source of error in any protein assay. The best choice for an antibody quantitation standard is a purified, known concentration of the specific immunoglobulin being tested. Often this is not available or it is too expensive to use as a standard. In such cases, the best standard is one that will produce a representative color response curve with the selected protein assay and is readily available to any researcher. BGG provides this function for all kinds of immunoglobulin samples, including IgG.

For greatest accuracy in estimating total protein concentration in unknown samples, it is essential to include a standard curve each time the assay is performed. This is particularly true for the protein assay methods that produce non-linear standard curves. Deciding on the number of standards and replicates used to define the standard curve depends upon the degree of non-linearity in the standard curve and the degree of accuracy required. In general, fewer points are needed to construct a standard curve if the color response is linear. Typically, standard curves are constructed using at least two replicates for each point on the curve.

For added safety when opening glass ampules, consider using our Ampule Breakers, which are disposable safety devices that protect the fingers when breaking open a glass ampule.

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Pierce™ Bovine Gamma Globulin Standard Pre-Diluted Set

Pierce™ Quantitative Peroxide Assay Kit (Lipid) (Thermo Scientific™)

The Thermo Scientific Pierce Quantitative Peroxide Assay Kit (lipid-compatible formulation) detects and measures hydrogen peroxide levels in biological samples using an iron and xylenol orange (XO) reagent.

This lipid-compatible peroxide assay kit detects peroxides based on oxidation of ferrous to ferric ion in the presence of xylenol orange. The kit's formulation may be used with lipid-containing samples without first extracting the lipids; however, because this kit does not contain sorbitol, the assay is less sensitive. Most proteins do not interfere with this assay, although some metal chelators may require use of a blank (i.e., control) to mitigate these effects.

Features of the Quantitative Peroxide Assay Kit:

Peroxides—detects and measures hydrogen peroxide (H2O2) levels in biological and other liquids samples
Convenient—no proprietary reagents involved but the ready-to-use kit removes the variability, difficulty and added expense of formulating the reagents yourself

Applications:
• General hydrogen peroxide (H2O2) measurement in protein samples
• Quantitation of peroxides in detergents
• Assessment of the effects of peroxides on cellular activity
• Determination of protein glycation; Measurement of peroxide accumulation in lipids

In this assay, hydroperoxides convert the Fe2+ to Fe3+ at acidic pH. With this lipid-compatible formulation, the peroxide converts the Fe2+ to Fe3+ directly. In a sulfuric acid solution, the Fe3+ complexes with the xylenol orange (XO) dye to yield a purple product having an absorbance maximum at 560nm. Peroxide levels in test samples can be determined by calculation from the known extinction coefficient of the XO-Fe complex or by reference to a standard curve prepared with hydrogen peroxide solution.

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Pierce™ Quantitative Peroxide Assay Kit (Aqueous)
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Pierce™ Streptavidin Coated Plate IP Kit (Thermo Scientific™)

The Thermo Scientific Pierce Streptavidin Coated Plate IP Kit uses coated streptavidin microplates to perform immunoprecipitation assays in 96-well plates without resins, beads, centrifugation or magnets.

Pierce Coated Plate IP Kits enable rapid immunoprecipitation of multiple samples without the usual tedium of pipetting, centrifuging and separating beaded affinity resin in individual microcentrifuge tubes. Immunoprecipitation is accomplished using coated 96-well microplates rather than beaded agarose resin. The plate format allows for faster processing of multiple samples.

Features of the Streptavidin Coated Plate IP Kit:

• Ready-to-use, high quality coated plates provide high capacity and consistency
• Plate format best suited for simultaneously processing multiple samples and their control conditions
• Faster, easier and more thorough washing than with traditional tube/resin IP methods
• Uses familiar and convenient ELISA tools (multichannel pipettors and plate washing); no tedious separation of supernatant from pelleted resin beads, and no tubes to open and close and centrifuge
• Coated plates are 96-well strip plates, convenient for experiments requiring only a partial plate
• Easy-to-follow instructions, including detailed explanation of appropriate controls

Applications:
• Rapid, trouble-free immunoprecipitation of multiple samples and their controls
• Immunoprecipitation when plate-based tools and techniques are preferred

Streptavidin is a protein that binds specifically and very strongly to biotin; therefore, the Streptavidin Coated Plate IP Kit (Part No. 45360) is appropriate for immunoprecipitation when using a biotin-labeled (biotinylated) antibody. In fact, this kit can be used to affinity purify a binding partner to any antibody species or subclass or any other protein or molecule that is biotinylated. Because the streptavidin-biotin affinity interaction is so strong, the elution step generally will dissociate only the antigen (binding partner), not the biotinylated antibody or "bait" protein.

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Pierce™ Bovine Gamma Globulin Standard Pre-Diluted Set (Thermo Scientific™)

Thermo Scientific Pierce BGG Protein Assay Standards are high-quality reference samples for generating accurate standard curves and calibration controls in total protein assays.

Features of BGG Protein Assay Standards:

Convenient—complete set containing seven ready-to-use dilutions
Antibody standard—best reference standard for immunoglobulin quantitation in colorimetric protein assays
Bradford standard—best general protein standard in coomassie-based (Bradford) protein assays
Pure and stable—supplied in ultrapure 0.9% saline solution with 0.05% sodium azide; room temperature stable
Accurate and consistent—precisely formulated at 2.00 +/-0.03 mg/mL, ensuring excellent lot-to-lot consistency

These bovine gamma globulin (BGG) solutions are protein concentration reference standards for use in BCA, Bradford and other protein assay protocols. BGG is an accepted reference protein for total protein quantitation of purified antibodies or immunoglobulin-rich samples. The IgG standard is precisely formulated at 2 mg/mL in an ultrapure 0.9% sodium chloride (saline) solution. The concentration of the stock solution of purified BGG (Fraction II) is calibrated by direct comparison to an internal standard to ensure lot-to-lot consistency and accuracy.

Applications:
• Protein assay immunoglobulin quantitation standard (Coomassie-Bradford Assay, etc.)
• Antibody recovery control for desalting and other column procedures
• Loading control for SDS-PAGE
• General calibration of spectrophotometer UV-lamp (absorbance at 280nm)

Selection of a protein standard is potentially the greatest source of error in any protein assay. The best choice for an antibody quantitation standard is a purified, known concentration of the specific immunoglobulin being tested. Often this is not available or it is too expensive to use as a standard. In such cases, the best standard is one that will produce a representative color response curve with the selected protein assay and is readily available to any researcher. BGG provides this function for all kinds of immunoglobulin samples, including IgG.

For greatest accuracy in estimating total protein concentration in unknown samples, it is essential to include a standard curve each time the assay is performed. This is particularly true for the protein assay methods that produce non-linear standard curves. Deciding on the number of standards and replicates used to define the standard curve depends upon the degree of non-linearity in the standard curve and the degree of accuracy required. In general, fewer points are needed to construct a standard curve if the color response is linear. Typically, standard curves are constructed using at least two replicates for each point on the curve.

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Pierce™ Bovine Gamma Globulin Standard Ampules, 2 mg/mL

Pierce™ Modified Lowry Protein Assay Kit (Thermo Scientific™)

The Thermo Scientific Pierce Modified Lowry Protein Assay is a stable form of a traditional, 2-component, folin phenol- and copper-based reagent system to measure (A750nm) total protein concentration compared to a protein standard.

Features of Modified Lowry Protein Assay:

Popular method—widely cited in protein research literature
Colorimetric—measure with a standard spectrophotometer or plate reader (750nm)
Stable—uses a modified cupric sulfate-tartrate reagent that is stable at room temperature
Large range—exhibits good linearity in the range 1 to 1500 µg/mL (tested with BSA protein)
Convenient—microplate and cuvette protocols provided with the instructions

The Modified Lowry Protein Assay Kit combines a stabilized formulation of the original Lowry Reagents and the essential Folin-Ciocalteu Phenol reagent in a complete kit for accurately determining protein concentration in a variety of samples types. Although newer protein assay methods provide greater speed and convenience, the Lowry method remains a popular, accurate and useful option for many applications.

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Pierce™ His Protein Interaction Pull-Down Kit (Thermo Scientific™)

Thermo Fisher Scientific Pierce His Tag Protein Interaction Pull-Down Kit contains the necessary components to capture and purify proteins that interact with His-tagged fusion proteins.

Features of His Tag Protein Interaction Pull-Down Kit:

6xHis pull-down (Product No. 21277)—purifies protein interactors of any His-tagged fusion protein
Complete kits—provide all components and detailed protocol for purifying protein:protein interactions
No special equipment needed—use common laboratory equipment and reagents (e.g., microcentrifuge)
Convenient—microcentrifuge spin columns facilitate simple and efficient manipulation of agarose beads, including simple processing of multiple samples
Flexible—instructions include protocols for use with bait and prey proteins expressed from a variety of sample types
Less non-specific binding—Cobalt chelate resin is more specific for histidine-tagged fusion proteins than nickel resins, resulting in less non-specific binding
Binding—Binds 10 to 25 mg of histidine-tagged fusion protein per mL of resin

Applications:

• Discover a new protein:protein interaction from a cell lysate
• Confirm a putative interaction from a cell lysate or with a previously purified protein
• Extract protein:protein interaction information from in vitro transcription/translation lysates

You provide the tagged fusion protein as the "bait" and the cells expressing the putative protein interaction target ("prey"), and the Pull-Down Kits provide everything else: cell lysis buffer, microcentrifuge spin columns, tag-specific affinity resin (agarose beads) and optimized buffers and protocol. The Pull-Down Kits are designed to teach the method to first-time users and to increase ease-of-use, convenience and reproducibility for experienced researchers.

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ProteinSEQ™ Protein A Core Kit (Applied Biosystems™)

The ProteinSEQ™ Protein A Core Kit is a partial kit that enables users to customize their ProteinSEQ protein measurements for leeched Protein A, or any other ligand used in their purification columns. It contains the buffers and reagents, but not the antibody-loaded capture beads, needed to enable PCR-based quantitation of Protein A or any other purification ligand. The kit contains sufficient reagents for 200 reactions. For a kit that includes Protein A capture beads and standards, for use in the quantitation of Protein A, please see the ProteinSEQ™ Protein A Quantitation Kit (Cat. No. 4469343).

• Quantitate leeched protein contaminants with ultra-high sensitivity
• Accelerate process development with more valid test results per run
• Reduce assay hands-on time with less sample dilution and preparation
• Achieve unprecedented productivity with near-5-log assay dynamic range

ProteinSEQ Protein A quantitation
The ProteinSEQ assay platform uses qPCR technology to measure process contaminants with extraordinary sensitivity and unparalleled dynamic range. Protein A or other purification ligands leeching from purification columns can be accurately measured with outstanding spike recovery and dilutional linearity.

Streamlined assay worflow
The ProteinSEQ system is the only instrument platform-based Protein A and purification ligand quantitation solution to offer single-digit picogram sensitivity and nearly 5 logs of dynamic range. Laboratory workflow is greatly accelerated due to the reduction in the number of sample dilutions and replicates in each run.

Pierce™ Bovine Serum Albumin Standard Pre-Diluted Set (Thermo Scientific™)

Thermo Scientific Pierce BSA Protein Assay Standards are high-quality reference samples for generating accurate standard curves and calibration controls in total protein assays.

Features of BSA Protein Assay Standards:

Convenient—complete set containing seven ready-to-use dilutions
Universal—recognized as the industry standard for protein quantitation in colorimetric protein assays
Pure and stable—supplied in ultrapure 0.9% saline solution with 0.05% sodium azide

These bovine serum albumin (BSA) solutions are protein concentration reference standards for use in BCA, Bradford and other protein assay protocols. BSA is the universally accepted reference protein for total protein quantitation. The set of standards is prepared from a stock solution that is calibrated by direct comparison to purified BSA (Fraction V) from the National Institute of Standards and Technology (NIST).

Applications:
• Protein assay quantitation standard (BCA Protein Assay, Coomassie-Bradford Assay, etc.)
• Protein recovery control for desalting and other column procedures
• General calibration of spectrophotometer UV-lamp (absorbance at 280nm)

Selection of a protein standard is potentially the greatest source of error in any protein assay. The best choice for a standard is a purified, known concentration of the most abundant protein contained in the samples being tested. Often, a highly purified, known concentration of the protein of interest is not available or it is too expensive to use as the standard, or the sample itself is a mixture of many proteins (e.g., cell lysate). In such cases, the best standard is one that will produce a normal (i.e., average) color response curve with the selected protein assay method and is readily available to any researcher. BSA is such a protein, and the Pierce Albumin Standards are the most convenient source of ready-to-use BSA standard.

For greatest accuracy in estimating total protein concentration in unknown samples, it is essential to include a standard curve each time the assay is performed. This is particularly true for the protein assay methods that produce non-linear standard curves. Deciding on the number of standards and replicates used to define the standard curve depends upon the degree of non-linearity in the standard curve and the degree of accuracy required. In general, fewer points are needed to construct a standard curve if the color response is linear. Typically, standard curves are constructed using at least two replicates for each point on the curve.

Related Products
Pierce™ Bovine Serum Albumin Standard Ampules, 2 mg/mL

Pierce™ Quantitative Peroxide Assay Kit (Aqueous) (Thermo Scientific™)

The Thermo Scientific Pierce Quantitative Peroxide Assay Kit (aqueous-compatible formulation) detects and measures hydrogen peroxide levels in biological samples using an iron and xylenol orange (XO) reagent.

This aqueous-compatible peroxide assay kit detects peroxides based on oxidation of ferrous to ferric ion in the presence of xylenol orange. The kit's formulation includes sorbitol, which provides sensitivity enhancement. Most proteins do not interfere with this assay, although some metal chelators may require use of a blank (i.e., control) to mitigate these effects. This kit provides for maximum sensitivity with non-lipid samples.

Features of the Quantitative Peroxide Assay Kit:

Peroxides—detects and measures hydrogen peroxide (H2O2) levels in biological and other liquids samples
Convenient—no proprietary reagents involved but the ready-to-use kit removes the variability, difficulty and added expense of formulating the reagents yourself

Applications:
• General hydrogen peroxide (H2O2) measurement in protein samples
• Quantitation of peroxides in detergents
• Assessment of the effects of peroxides on cellular activity
• Determination of protein glycation; Measurement of peroxide accumulation in lipids

In this assay, hydroperoxides convert the Fe2+ to Fe3+ at acidic pH. With this aqueous-compatible formulation, peroxide first reacts with sorbitol, converting it to a peroxyl radical, which in turn initiates Fe2+ oxidation to Fe3+. In a sulfuric acid solution, the Fe3+ complexes with the xylenol orange (XO) dye to yield a purple product having an absorbance maximum at 560nm. Peroxide levels in test samples can be determined by calculation from the known extinction coefficient of the XO-Fe complex or by reference to a standard curve prepared with hydrogen peroxide solution.

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Pierce™ Peroxide Assay Reagent B
Pierce™ Peroxide Assay Reagent C

Compat-Able™ Protein Assay Preparation Reagent Kit (Thermo Scientific™)

The Thermo Scientific Compat-Able Protein Assay Preparation Set eliminates interfering substances from protein assay samples to make them compatible with BCA and Bradford assays.

The Compat-Able Reagents remove salts, detergents, reducing agents and other substances from protein samples to eliminate interference with protein assays. The protocol selectively precipitates all of the protein, thereby allowing the non-protein components to be easily decanted and removed. The purified, precipitated protein is redissolved in water, making it ready for accurate determination of the protein concentration by standard protein assay methods. The procedure is accomplished in less than 10 minutes with just a few simple steps. The Compat-Able Preparation Set is available individually or conveniently bundled with either the Thermo Scientific Pierce BCA Protein Assay Kit or the Thermo Scientific Pierce Coomassie Plus Protein Assay Kit.

Features of the Compat-Able Protein Assay Preparation Reagent Kit:

Eliminate incompatibilities—removes nearly any substance that interferes with colorimetric protein assays: salts, detergents, denaturants, reducing agents, and even IEF sample buffer
Simple and fast—prepare samples in less than 10 minutes with four simple steps
Convenient—reagent set is easier to use than homemade TCA or acetone precipitation reagents, thereby producing more consistent results
Scalable—microcentrifuge method is easily scaled to larger centrifuge tube volumes for special needs
Validated—tested and offered together with BCA and Coomassie Plus Protein Assay Kits; protocol integrates control and normalization procedures

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Pierce™ Detergent Compatible Bradford Assay Kit (Thermo Scientific™)

The Thermo Scientific™ Pierce™ Detergent Compatible Bradford Assay Kit is a quick and ready-to-use modification of the well-known Bradford coomassie dye-binding, colorimetric method for total protein quantitation. Proprietary additives to the Bradford Reagent make it compatible with up to 1% or higher of detergents and lysis reagents that are commonly used in life science research, including Triton® X-100 and NP-40.

• Convenient— detergent-free standard curve
• Flexible— compatible with samples both with and without detergent
• Minimal sample—requires only 10 µL for microplate procedure
• Colorimetric—measure with a standard spectrophotometer or plate reader (595 nm)
• Easy to use— single reagent, no working reagent preparation required
• Fast—10 minute incubation at room temperature
• Broad range—detects protein concentration in the ranges of 2 to 1500 µg/mL

Similar to the Bradford method, coomassie dye binds protein in an acidic medium causing an immediate shift in absorption maximum from 465 nm to 595 nm with a concomitant color change from green to blue. In addition, the assay is complete in just 10 minutes.

The protein assay can be performed in either test tube or microplate format. The standard working range is 100-1500 µg/mL with up to 1% detergent (or higher in some cases). Protein concentrations are estimated by reference to absorbances obtained for a series of standard protein dilutions, typically bovine serum albumin (BSA), which are assayed alongside the unknown samples. Because the color response with coomassie dye is non-linear with increasing protein concentration, a standard curve must be completed with each assay. Standards can be used directly without preparing them in the same detergent found in the test samples.

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Ampule Breakers