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

The Thermo Scientific Pierce Coomassie Plus Protein Assay is a ready-to-use, reducing agent-compatible, improved Bradford assay reagent to quickly measure (A595nm) total protein concentration compared to a protein standard.

The Pierce Coomassie Plus Assay Reagent is a single, ready-to-use solution for measuring protein concentration. Simply add the reagent to equal volumes of samples and standards, mix and then measure the absorbance at 595nm. The assay costs only pennies per sample and can be performed in either test tube or microplate format. The Pierce Coomassie Plus Assay Reagent provides increased linearity of response and only half the protein-to-protein variability of other commercial Bradford assay formulations.

Features of the Coomassie Plus Protein Assay:

Colorimetric—measure with a standard spectrophotometer or plate reader (595nm)
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
Better—improved linearity and response-uniformity compared to traditional Bradford formulations
Flexible—microplate and cuvette protocols provided with the instructions and adaptable to several target working ranges

How the Coomassie Plus (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 610nm). The difference between the two forms of the dye is greatest at 595nm, 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 575nm and 615nm. At the two extremes (575nm and 615nm) there is a loss of about 10% in the measured amount of color (absorbance) compared to that obtained at 595nm.

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 3,000 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 595nm 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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Pierce™ Coomassie Plus (Bradford) Assay Reagent
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Ampule Breakers

Micro BCA™ Reagent B (MB) (Thermo Scientific™)

Thermo Scientific Pierce Micro BCA Reagent B (MB) is a proprietary bicinchonic acid solution. This product is sufficient for 3200 microplate assays when mixed with Reagents MA and MC.

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Micro BCA™ Protein Assay Kit
Micro BCA™ Reagent A (MA)
Micro BCA™ Reagent C (MC)

Pro-Detect™ Rapid DYKDDDDK-His Assay Kit (Thermo Scientific™)

The Thermo Scientific Pro-Detect Rapid DYKDDDDK-His Assay Kit is a single-use, dipstick lateral flow kit for quick, easy detection of DYKDDDDK-His double-tagged proteins in protein expression and purification systems. This lateral flow assay uses membrane-based strips coated with dye-bound conjugated antibodies specific to the DYKDDDDK-His tag to provide a visual, color readout for detection within ten minutes. This assay can be used to detect DYKDDDDK-His-tagged proteins directly from cell culture media (supernatant) or cell lysates without any special instrumentation or handling.

Features of the Pro-Detect Rapid assay kits:
Easy to adopt—lateral flow strips can be used early in and throughout expression to monitor and confirm expression of desired protein-tagged target
Simple, fast method—simply dilute sample using provided diluent and dip in the lateral flow strip for visual results within 5-10 minutes
Sensitive—detect protein tags in supernatant or cell lysate samples down to low µg/mL concentrations
Specific—assay does not cross-react with common lysis and protein preparation reagents
Reliable—results are similar to other antibody immunoassays, including western blot and ELISA
Long shelf life—stable for at least one year at 4°C

Each Pro-Detect Rapid assay is performed by simply adding a properly diluted tissue culture supernatant, lysate, or purified protein sample to a test tube or microplate well and dipping in a lateral flow strip. Gold-conjugated detection antibodies embedded in the strip form complexes with the tagged protein in the sample. These complexes travel the length of the strip membrane and bind at the test and control lines. Results are displayed as red bands.

Pierce™ c-Myc-Tag IP/Co-IP Kit (Thermo Scientific™)

The Thermo Scientific Pierce c-Myc Tag IP/Co-IP Kit provides the affinity resin and other reagents necessary to easily perform immunoprecipitation (IP) or co-immunoprecipitation (co-IP) experiments using a c-Myc-tagged protein as the bait.

The c-Myc peptide (EQKLISEEDL) has become a popular fusion tag for mammalian recombinant protein expression. This kit includes crosslinked beaded agarose to which a highly specific anti-c-Myc antibody is covalently immobilized. Upon incubation with a sample containing the tagged fusion protein, interaction complexes involving the c-Myc-tagged bait protein are captured on the agarose beads. After simple washing steps, the specific protein interaction complex is easily eluted from the resin in the supplied elution buffer or SDS-PAGE sample loading buffer for subsequent analysis. The kit includes the prepared agarose affinity resin, buffers, microcentrifuge spin columns, a positive control and easy-to-follow instructions.

Features of the c-Myc-Tag IP/Co-IP Kit:

Specific—the immobilized anti-c-Myc monoclonal antibody binds the c-Myc epitope tag with high specificity, providing high yield immunoprecipitation products and clean Western blot detection
High capacity—excellent results with as little as 1 µg of anti-c-Myc antibody in IP mode with the positive control lysate
Rigorous—kit includes a positive control lysate that contains over-expressed GST-c-Myc to validate the reagents and specific protocol, and the instructions provide tips for planning other controls
Robust—the affinity system is compatible with IP or Co-IP from various cell lysates and physiological (non-denaturing) buffer systems
Convenient and easy—complete kit includes all necessary reagents, convenient spin columns, and easy-to-follow instructions

The c-Myc peptide (EQKLISEEDL) derived from the C-terminus region of human c-Myc protein is one of several fusion protein tags used for recombinant protein expression. Utilizing a specific, high-affinity immobilized antibody, c-Myc tagged fusion proteins can be quickly purified from bacterial and mammalian cell lysates as well as from the Pierce Human in vitro translation reactions. For co-immunoprecipitation reactions, simple wash steps allow enrichment and elution of specific protein interaction complexes into the supplied elution buffer or SDS-PAGE sample loading buffer for subsequent analysis.

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CBQCA Protein Quantitation Kit (Invitrogen™)

The CBQCA Protein Quantitation Kit is a very sensitive assay for quantitating proteins in solution, capable of detection as low as 10 ng of protein per mL. Similar in sensitivity to our NanoOrange protein quantitation reagent (N-6666), CBQCA is better suited for accurate quantitation of proteins in the presence of lipids, membrane fractions or detergents, and for lipoproteins and small peptides.

Pro-Detect™ Rapid MBP Assay Kit (Thermo Scientific™)

The Thermo Scientific Pro-Detect Rapid MBP Assay Kit is a single-use, dipstick lateral flow kit for quick, easy detection of maltose binding protein (MBP)-tagged proteins in protein expression and purification systems. This lateral flow assay uses membrane-based strips coated with dye-bound conjugated antibodies specific to the MBP tag to provide a visual, color readout for detection within ten minutes. This assay can be used to detect MBP-tagged proteins directly from cell culture media (supernatant) or cell lysates without any special instrumentation or handling.

Features of the Pro-Detect Rapid assay kits:
Easy to adopt—lateral flow strips can be used early in and throughout expression to monitor and confirm expression of desired protein-tagged target
Simple, fast method—simply dilute sample using provided diluent and dip in the lateral flow strip for visual results within 5-10 minutes
Sensitive—detect protein tags in supernatant or cell lysate samples down to low µg/mL concentrations
Specific—assay does not cross-react with common lysis and protein preparation reagents
Reliable—results are similar to other antibody immunoassays, including western blot and ELISA
Long shelf life—stable for at least one year at 4°C

Each Pro-Detect Rapid assay is performed by simply adding a properly diluted tissue culture supernatant, lysate, or purified protein sample to a test tube or microplate well and dipping in a lateral flow strip. Gold-conjugated detection antibodies embedded in the strip form complexes with the tagged protein in the sample. These complexes travel the length of the strip membrane and bind at the test and control lines. Results are displayed as red bands.

Pierce™ Rapid Gold BCA Protein Assay Kit (Thermo Scientific™)

The Thermo Scientific Pierce Rapid Gold BCA Protein Assay Kit is a two-component, high-precision, detergent-compatible assay optimized to measure (at 480 nm) total protein concentration compared to a protein standard curve of known concentrations. The Pierce Rapid Gold BCA Protein Assay uses the same copper-chelating technology as the well-known traditional Pierce BCA Protein Assay and provides comparable accuracy but with a 5 minute, room temperature incubation and measured at 480 nm. This improvement eliminates the need to wait or expose the samples to elevated temperatures for a fast time to results.

Features of the Rapid Gold BCA Protein Assay Kit:
• Colorimetric—estimate visually or measure with a standard spectrophotometer or plate reader (480 nm)
• Excellent uniformity—exhibits similar protein-to-protein variation as traditional BCA Protein Assay, and less protein-to-protein variation than dye-binding protein assay methods
• Compatible—unaffected by typical concentrations of most ionic and nonionic detergents
• Fast time to results—significant improvement over traditional BCA Assay with a 5 minute, room temperature reaction
• High linearity—linear working range for BSA equals 20 to 2000 µg/mL
• Optimal absorbance on standard spectrophotometers—reaction produces a color change to an intense orange-gold that results in a strong linear response at 480 nm

The traditional BCA Protein Assay is a widely used and accepted protein assay, trusted for its highly accurate protein concentration determination and compatibility with most sample types encountered in protein research. However, in order to develop the samples in this traditional protocol, one must either heat the reaction at 37°C for 30 minutes or allow room temperature development for 2 hours. The Rapid Gold BCA Protein Assay maintains the key characteristics of the traditional BCA assay but allows a fast time and room temperature incubation equal to dye-binding methods. The Rapid Gold BCA assay can be used to assess yields in whole cell lysates and affinity-column fractions, as well as to monitor protein contamination in industrial applications. Compared to most dye-binding methods and similar to the traditional BCA assay, the Rapid Gold BCA assay is affected much less by protein compositional differences, providing low protein-to-protein variation.

Applications:
• Studying protein:protein interactions
• Measuring column fractions after affinity chromatography
• Estimating percent recovery of membrane proteins from cell extracts
• High-throughput screening of fusion proteins

How the Rapid Gold BCA Protein Assay detects protein
The Rapid Gold BCA Protein Assay uses the same copper reduction method as the traditional BCA Protein Assay with a proprietary copper chelator. The Rapid Gold BCA assay combines the well-known reduction of Cu2+ to Cu1+ by protein in an alkaline medium with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu1+) by the proprietary chelator. The first step is the chelation of copper with protein in an alkaline environment to form a light green complex. In this reaction, known as the biuret reaction, peptides containing three or more amino acid residues form a colored chelate complex with cupric ions in an alkaline environment containing sodium potassium tartrate.

In the second step of the color development reaction, the Rapid Gold BCA chelator reacts with the reduced (cuprous) cation that was formed in step one. The intense gold-colored reaction product results from the chelation of two molecules of BCA with one cuprous ion. The BCA/copper complex is water-soluble and exhibits a strong linear absorbance at 480 nm with increasing protein concentrations. The BCA reagent is approximately 100 times more sensitive (lower limit of detection) than the pale green color of the first reaction.

The reaction that leads to the color formation is strongly influenced by four amino acid residues (cysteine or cystine, tyrosine, and tryptophan) in the amino acid sequence of the protein. However, unlike the Coomassie dye-binding methods, the universal peptide backbone also contributes to color formation, helping to minimize variability caused by protein compositional differences.

Compatibility of the Rapid Gold BCA Protein Assay
Certain substances are known to interfere with the Rapid Gold BCA Protein Assay including those with reducing potential, chelating agents, and strong acids or bases. Because they are known to interfere with protein estimation at even minute concentrations, avoid the following substances as components of the sample buffer:
• Ascorbic acid
• EGTA
• Iron
• Impure sucrose
• Catecholamines
• Impure glycerol
• Lipids
• Tryptophan
• Creatinine
• Hydrogen peroxide
• Melibiose
• Tyrosine
• Cysteine
• Hydrazides
• Phenol Red
• Uric acid

Protein assays using a copper chelator are found to be highly advantageous in that most surfactants (even if present in the sample at concentrations up to 5%) are compatible with the assay. Thermo Fisher Scientific has performed extensive compatibility experiments on all of our protein assays, in particular for the traditional BCA Protein Assay. The new Rapid Gold BCA Protein Assay was determined to have similar compatibility of interfering substances as traditional BCA. Additional data and information can be found in the instructional booklet for each protein assay.

Pro-Detect™ Rapid DYKDDDDK Competitive Assay Kit (Thermo Scientific™)

The Thermo Scientific Pro-Detect Rapid DYKDDDDK Competitive Assay Kit is a single-use, dipstick lateral flow kit for quick, easy detection of DYKDDDDK (commonly known as "FLAG")-tagged proteins in protein expression and purification systems. This lateral flow assay uses membrane-based strips coated with dye-bound conjugated antibodies specific to DYKDDDDK tag to provide a qualitative detection within ten minutes. This assay can be used to detect DYKDDDDK-tagged proteins directly from cell culture media (supernatant) or cell lysates without any special instrumentation or handling.

Features of the Pro-Detect Rapid competitive assay kits:
Easy to adopt—lateral flow strips can be used early in and throughout expression to monitor and confirm expression of desired protein-tagged target
Simple, fast method—simply dilute sample using provided diluent and dip in the lateral flow strip for qualitative results within 5-10 minutes
Sensitive—detect protein tags in supernatant or cell lysate samples down to low µg/mL concentrations
Specific—assay does not cross-react with common lysis and protein preparation reagents
Reliable—results are similar to other antibody immunoassays, including western blot and ELISA
Long shelf life—stable for at least one year at 4°C

Each Pro-Detect Rapid competitive assay is performed by simply adding a properly diluted tissue culture supernatant, lysate, or purified protein sample to a test tube or microplate well and dipping in a lateral flow strip. Gold-conjugated detection antibodies embedded in the strip form complexes with the tagged protein in the sample. In the presence of target protein tag, the dye-bound conjugated antibodies will bind to the tagged protein present in the sample instead of the protein tag bound at the test lines. Therefore, in a positive result, a competitive lateral flow assay will show decreasing intensity of the visible test lines until all test lines have disappeared. When there is no target protein tag present, the dye-bound conjugated antibodies specific to the target will bind at the test line(s), resulting in visual test lines in a negative result.

Pierce™ Direct IP Kit (Thermo Scientific™)

The Thermo Scientific Pierce Direct IP Kit uses an activated resin to covalently immobilize IP antibodies (any species or class) on agarose beads without the aid of Protein A/G, enabling immunoprecipitation without antibody interference.

The primary benefits resulting from this method are the opportunity to use any species or subclass of purified antibody (not just types that bind to Protein A or G) and the ability to purify target protein without contamination by the antibody. The method also makes it possible to immunoprecipitate antigens from serum samples without co-purifying non-target immunoglobulins. Finally, the kit uses microcentrifuge spin cups to effectively wash and separate samples from the beaded agarose resin.

Features of Direct IP Kit:

Requires less than 10 µg of antibody—optimized procedure requires only 2 to 10 µg of antibody for a single IP experiment, no more than is required for traditional IP methods
Easy and efficient scalability—use only the amount of antibody needed for a single immunoprecipitation or immobilize 100 to 200 µg of antibody to prepare ready-made IP affinity resin for many experiments
Minimal antibody contamination—antibody is irreversibly attached to the agarose beads so that co-elution of heavy and light chains with the purified protein is minimized (i.e., less than 5%)
IP with any species and subclass of antibody—Use chicken IgY, human IgE, mouse IgM or any other purified protein
Prepared antibody affinity resin is reusable—because the antibody is covalently immobilized and not inactivated by the mild elution procedure, the resin often can be used several times
Convenient sample handling—spin columns (see dimensions) eliminate resin loss and provide for efficient separation of solutions
Complete kit—package includes cell lysis buffer and sufficient reagents and spin columns to perform at least 50 antibody immobilizations and IP experiments
Requires purified antibody—IP antibody solution must be free of BSA, gelatin or other stabilizer proteins; for easy removal of these proteins, see our Antibody Clean-up Kit (Part No. 44600)

Applications:
• Immunoprecipitation followed by electrophoresis and excision of the protein for mass spectrometry or sequencing identification analysis
• Immunoprecipitation and SDS-PAGE analysis of a target protein whose molecular weight is similar to the heavy or light chain antibody fragment
• Immunoprecipitation of target proteins for subsequent analysis by nondenaturing methods (i.e., methods not involving SDS-PAGE)
• Immunoprecipitation with antibodies that do not bind to Protein A, Protein G or Protein A/G
• Immunoprecipitation from serum and other sample that contain immunoglobulins

Direct immunoprecipitation in two easy steps:

Step 1: Attach antibody to agarose resin: The first step in the Direct IP Method is to prepare the IP antibody affinity resin. Pure antibody (see Highlights) is incubated in phosphate buffer with an appropriate volume of AminoLink Plus Coupling Resin, an aldehyde-activated beaded agarose resin. During the incubation, various lysine epsilon-amines on the surface of the large antibody molecule react with aldehyde groups on the resin to form Schiff-base bonds that are then stabilized to secondary amine bonds in the presence of cyanoborohydride. The immobilization reaction, known as reductive amination, is nondenaturing and typically results in greater than 85% antibody coupling efficiency with minimal loss of antigen-binding function.

Step 2: Perform immunoprecipitation experiments: Once prepared, the antibody resin can be used in several parallel aliquots or in total for IP experiments. Antibody resin is incubated with a sample that contains the protein antigen of interest, allowing the antibody:antigen complex to form. After washing to remove nonbound (presumably undesired) components of the sample, the antigen is recovered by dissociation from the antibody with elution buffer supplied in the kit. The entire procedure is performed in a microcentrifuge spin cup, allowing solutions to be fully separated from the agarose resin upon brief centrifugation. Only antigen is eluted by the procedure, enabling it to be identified and further analyzed without interference from antibody fragments. Furthermore, the antibody resin can be reused for additional rounds of immunoprecipitation.

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Pierce™ Direct Magnetic IP/Co-IP Kit

Pierce™ Direct Magnetic IP/Co-IP Kit (Thermo Scientific™)

Thermo Scientific Pierce Direct Magnetic IP/Co-IP Kit uses NHS-chemistry to covalently immobilize IP antibodies onto magnetic beads for effective immunoprecipitation and co-immunoprecipitation.

Magnetic IP with NHS chemistry is convenient and allows for the immobilization of IP antibody from any species and subtype without the variations in coupling efficiency seen with Protein A and Protein G. The IP antibody is covalently attached so target proteins or co-IP protein complexes can be eluted and analyzed without antibody contamination. The kit contains an optimized protocol and all buffers and reagents necessary for antibody coupling and high yield IP or co-IP using either manual magnetic stands or automated platforms such as the Thermo Scientific KingFisher Instruments.

Features of the Direct Magnetic IP/Co-IP Kit:

Antibody-free IP—antibody is irreversibly attached to the magnetic beads, resulting in negligible co-elution of intact antibody or heavy and light chains with the purified antigen
Fast—complete antibody conjugation and immunoprecipitation in less than 4 hours
Convenient—the IP/co-IP kit contains magnetic beads and all essential buffers for optimal immunoprecipitation
Antibody compatible—use with any antibody species, class or isotype
Low nonspecific binding—the bead surface is pre-blocked and all nonreacted NHS-ester groups are fully quenched
Protocol-compatible—protein coupling to the beads and immunoprecipitation can be performed manually or by automation (e.g., Thermo Scientific KingFisher Instruments)
Scalable—use only the amount of antibody needed for a single IP experiment or prepare a larger quantity of antibody magnetic beads for multiple experiments

The protocol for the Pierce Direct IP/Co-IP Kit conjugates IP antibody to Pierce NHS-Activated Magnetic Beads through NHS-ester chemistry. The antibody-bound beads are then incubated with quenching buffer to block remaining active NHS sites. The prepared antibody-bound beads are incubated with cell lysate or tissue extract that contain the protein antigen of interest, allowing the antigen:antibody complex to form. The beads are washed to remove nonbound material and a low-pH elution buffer is used to dissociate bound antigen from the antibody-bound bead. Neutralization buffer is included to prevent precipitation of the isolated antigen and to ensure protein activity in downstream applications. Lane Marker Sample Buffer is included for preparing samples for SDS-PAGE analysis. The protocol is optimized for 2 to 10μg of IP antibody per assay. For optimal co-IP yields, use 10μg of antibody. The beads can be processed manually using a magnetic stand or by automation with an instrument such as the Thermo Scientific KingFisher Flex.

Traditional IP with Protein A/G Magnetic Beads, involving passive, noncovalent coupling of antibody with Protein A/G typically provides higher antigen yields than IP protocols that involve covalent attachment of antibody to magnetic beads through crosslinking or NHS-ester chemistry. Covalent attachment inevitably causes loss of some functional antibody binding sites. To overcome this limitation with NHS-chemistry, it may be necessary to use larger amounts of IP antibody in the direct IP method than in the traditional IP method. The advantage of immunoprecipitation with IP antibody covalently bound to the magnetic bead is Western blots and gels devoid of interfering antibody bands.

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Pierce™ BCA Protein Assay Kit - Reducing Agent Compatible (Thermo Scientific™)

This BCA Protein Assay Kit is the reducing agent-compatible version of our popular Thermo Scientific Pierce BCA Protein Assay. The kit enables you to measure protein concentration in samples that contain thiol-reductants dithiothreitol (DTT) and 2-mercaptoethanol (BME).

Features of the BCA Protein Assay Kit—Reducing Agent Compatible:

Compatible—assay samples that contain up to 5 mM DTT, 35 mM BME, or 10 mM TCEP
BCA technology—only a slight modification of the standard BCA Protein Assay protocol (15-minute incubation with Compatibility Reagent); no precipitation steps required
Small samples—requires only 25 µL (standard kit) or less than 10 µL (microplate kit) of sample
Colorimetric—measure with a standard spectrophotometer or plate reader (562nm)
Excellent uniformity—exhibits less protein-to-protein variation than dye-binding methods
High linearity—linear working range for BSA equals 125 to 2000 µg/mL

The BCA Protein Assay Kit—Reducing Agent Compatible (BCA-RAC) provides all of the advantages of the original BCA Assay, plus compatibility with disulfide reducing agents at concentrations routinely used in protein sample buffers. This special adaptation of the popular Pierce BCA Protein Assay method enables accurate protein concentration measurement for samples containing DTT, 2-ME or TCEP. This reducing agent compatible (RAC) BCA Kit extends the already broad reagent compatibility of the BCA Protein Assay protocol to include nearly all types of components commonly present in protein research samples.

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Pierce™ Bovine Serum Albumin Standard Ampules, 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—1 mL ampules
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.

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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Pro-Detect™ Rapid V5 Competitive Assay Kit (Thermo Scientific™)

The Thermo Scientific Pro-Detect Rapid V5 Competitive Assay Kit is a single-use, dipstick lateral flow kit for quick, easy detection of V5-tagged proteins in protein expression and purification systems. This lateral flow assay uses membrane-based strips coated with dye-bound conjugated antibodies specific to V5 tag to provide a qualitative detection within ten minutes. This assay can be used to detect V5-tagged proteins directly from cell culture media (supernatant) or cell lysates without any special instrumentation or handling.

Features of the Pro-Detect Rapid competitive assay kits:
Easy to adopt—lateral flow strips can be used early in and throughout expression to monitor and confirm expression of desired protein-tagged target
Simple, fast method—simply dilute sample using provided diluent and dip in the lateral flow strip for qualitative results within 5-10 minutes
Sensitive—detect protein tags in supernatant or cell lysate samples down to low µg/mL concentrations
Specific—assay does not cross-react with common lysis and protein preparation reagents
Reliable—results are similar to other antibody immunoassays, including western blot and ELISA
Long shelf life—stable for at least one year at 4°C

Each Pro-Detect Rapid competitive assay is performed by simply adding a properly diluted tissue culture supernatant, lysate, or purified protein sample to a test tube or microplate well and dipping in a lateral flow strip. Gold-conjugated detection antibodies embedded in the strip form complexes with the tagged protein in the sample. In the presence of target protein tag, the dye-bound conjugated antibodies will bind to the tagged protein present in the sample instead of the protein tag bound at the test lines. Therefore, in a positive result, a competitive lateral flow assay will show decreasing intensity of the visible test lines until all test lines have disappeared. When there is no target protein tag present, the dye-bound conjugated antibodies specific to the target will bind at the test line(s), resulting in visual test lines in a negative result.

Pierce™ 660nm Protein Assay Reagent (Thermo Scientific™)

The Thermo Scientific Pierce 660 nm Protein Assay is a ready-to-use, detergent- and reducing agent-compatible assay reagent to quickly measure (A660 nm) total protein concentration compared to a protein standard.

The Pierce 660 nm Assay is more linear than coomassie-based Bradford assays and compatible with higher concentrations of most detergents, reducing agents and other commonly used reagents. The accessory Ionic Detergent Compatibility Reagent (IDCR) provides for even broader detergent compatibility, making this one of the only protein assays that is suitable for samples containing Laemmli SDS sample buffer with bromophenol blue. Although the Pierce 660 nm Protein Assay produces a higher level of protein-to-protein variation (37%) than other assays, such as the BCA Protein Assay, the simpler single-reagent format and broader substance compatibility make the Pierce 660 nm Assay more convenient for many routine applications. The Pierce 660 nm Protein Assay can be performed in either a test tube or microplate format.

Features of the 660 nm Protein Assay Reagent:

Versatile—works with a greater range of detergents and reducing agents than other dye-based assays
Fast—single reagent with a simple mix-and-read protocol; no working reagent to prepare
Accurate—produces standard curves that are more linear than with the Bradford assay method
Flexible—assay may be performed in test tubes or microplates
Conserve samples—requires only 10 µL for microplate or 100 µL for the test tube procedures
Convenient—room temperature storage means no waiting for reagent equilibration before use

How the Pierce 660nm Assay Detects Protein:
The Pierce 660nm Protein Assay is based on the binding of a proprietary dye-metal complex to protein in acidic conditions that causes a shift in the dye's absorption maximum, which is measured at 660nm. The dye-metal complex is reddish-brown and changes to green upon protein binding. The color change is produced by deprotonation of the dye at low pH facilitated by interactions with positively charged amino acid groups in proteins. Therefore, the dye interacts mainly with basic residues in proteins, such as histidine, arginine and lysine and to a lesser extent tyrosine, tryptophan and phenylalanine.

The color produced in the assay is stable and increases in proportion to a broad range of increasing protein concentrations, even in the presence of detergents and reducing agents that would be incompatible with Bradford and BCA Protein Assays. The optional IDCR may be added to the assay reagent to increase compatibility with high amounts of ionic detergents, allowing samples containing Laemmli SDS sample buffer with bromophenol blue to be measured. The IDCR completely dissolves by thorough mixing and does not have any effect on the assay.

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

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ProteinSEQ™ CHO HCP Quantitation Kit (Applied Biosystems™)

The ProteinSEQ™ CHO HCP Quantitation Kit is an assay kit containing all reagents and buffers needed to perform 200 PCR reactions to quantify process-independent host cell proteins from CHO cell lines. It combines a broadly reactive anti-CHO HCP antibody with a qPCR-based TaqMan™ Assay platform to enable measurements of HCPs with high sensitivity and wide dynamic range.

The ProteinSEQ CHO HCP Quantitation Kit contains:

• Capture beads loaded with anti-CHO HCP antibody
• CHO HCP standards
• All reagents and buffers needed to perform 200 PCR reactions for the process-independent quantitation of host cell proteins

Features include:

• Precise—quantitate host cell protein contaminants with excellent precision
• Fast—helps accelerate process development with more valid test results per run
• Efficient—helps reduce hands-on time with less sample dilution and preparation
• Reproducible—achieve unprecedented productivity with 4-log dynamic range

ProteinSEQ CHO HCP quantitation
The ProteinSEQ assay platform uses qPCR technology to measure protein process contaminants with extraordinary sensitivity and unparalleled dynamic range. Host cell protein contaminants from CHO cell lines can be accurately measured from 0.5–3,150 ng/mL, with outstanding quantitation efficiency and dilutional linearity.

Streamlined workflow
The ProteinSEQ CHO HCP Quantitation Kit is the only instrument platform-based HCP quantitation solution to deliver a generic, process-independent HCP assay with class-leading sensitivity and 4-log dynamic range. The kit helps accelerate laboratory workflows because the number of sample dilutions and replicates is reduced in each run.