The BrdU Cell Proliferation Assay Kit detects 5-bromo-2’-deoxyuridine (BrdU) incorporated into cellular DNA during cell proliferation using an anti-BrdU antibody. When cells are cultured with labeling medium that contains BrdU, this pyrimidine analog is incorporated in place of thymidine into the newly synthesized DNA of proliferating cells. After removing labeling medium, cells are fixed and the DNA is denatured with our fixing/denaturing solution. Denaturing of DNA is necessary to improve the accessibility of the incorporated BrdU to the detection antibody. A BrdU mouse mAb is then added to detect the incorporated BrdU. Anti-mouse IgG, HRP-linked Antibody is used to recognize the bound detection antibody. Chemiluminescent reagent is added for signal development. The magnitude of light emission, measured in relative light units (RLU), is proportional to the quantity of BrdU incorporated into cells, which is a direct indication of cell proliferation.
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Background: Halogenated nucleotides such as the pyrimidine analog bromodeoxyuridine (BrdU) are useful for labeling nascent DNA in living cells and tissues. BrdU becomes incorporated into replicating DNA in place of thymidine and subsequent immunodetection of BrdU using specific monoclonal antibodies allows labeling of cells in S phase of the cell cycle. After pulse-labeling cells or tissues with bromodeoxyuridine, BrdU (Bu20a) Mouse mAb can be used to detect BrdU incorporated into single stranded DNA. Please see our detailed protocol for information regarding the labeling procedure and denaturation of double stranded DNA for various immunodetection applications (1-4).
EDTA (Ethylenediaminetetraacetic acid) is a common laboratory chelating agent of divalent cations, such as Ca2+ and Mg2+. Ultrapure 0.5 M EDTA, pH 8.0 from Cell Signaling Technology contains no detectable DNase, RNase, or protease activity. The convenient 1 ml vials reduce the likelihood of contamination that can occur with larger volume containers. It is suitable for use in molecular biology or protein biochemistry applications that require the chelation of divalent metal cations.This product is used in our SimpleChip® chromatin immunoprecipitation (ChIP) assays to stop the metal-dependant enzymatic digestion of cross-linked DNA by micrococcal nuclease once the reaction is complete. It can be added to cell lysis buffers for use as a metalloprotease inhibitor. Working concentrations are typically 1-5 mM in this application.
Premium 16% (w/v) Formaldehyde from Cell Signaling Technology is used as a fixative agent for fluorescent immunocytochemical and flow cytometry assays. It is methanol-free, prepared from high quality paraformaldehyde, and packaged under an inert atmosphere of nitrogen.Each 10 ml solution is supplied in an amber glass vial with two access points, offering distinct advantages over pre-scored ampules. The screw cap allows for easy access to large volumes if necessary. To extend the product's shelf life, small volumes should be extracted by piercing the silicone top with a needle and syringe.
4% (w/v) Formaldehyde from Cell Signaling Technology is a ready-to-use fixative agent for fluorescent immunocytochemical and flow cytometry assays. It is formulated in 1X PBS, methanol-free, prepared from high-quality paraformaldehyde, and packaged under an inert atmosphere of nitrogen.
Ammonium bicarbonate buffer is supplied as a one molar (1M) concentrated stock in HPLC grade water. This buffer is recommended for use in PTMScan protocols for LC-MS applications during the digestion procedures.
Animal-Free Blocking Solution (5X) is designed for use as a blocking reagent in chromogenic immunohistochemical assays (IHC) and western blotting. This plant-based product contains no animal-derived proteins and can be used to decrease background in place of normal goat serum, BSA, or non-fat dry milk.
BackDrop® Green Background Suppressor is a novel reagent designed to effectively suppress green background fluorescence and improve live cell image quality, eliminating the need for medium removal and potential cell loss. BackDrop® Green is supplied as a ready-to-use liquid in a convenient dropper bottle and is applied directly to live cell imaging samples.
Ultra pure bovine serum albumin (BSA) is used as a blocking agent during Western blotting to reduce background and non-specific binding. BSA can also be used in other protocols that involve antibody binding including ELISA, DELFIA, flow cytometry and immunofluorescence/immunocytochemistry.
Dithiothreitol (DTT) from Cell Signaling Technology is offered in a convenient 192.8 mg lyophilized format, allowing for preparation of a fresh stock solution. This DTT reagent contains no detectable DNase or RNase activity and is suitable for use in molecular biology or protein biochemistry applications that require reduction of protein disulfide bonds.SDS-PAGE sample buffers are routinely supplemented with 10-50 mM DTT to cleave protein disulfide bonds. Lower concentrations of DTT are routinely used to stabilize enzymes or other proteins that posses free sulfhydryl groups, which is useful in chromatin immunoprecipitation (ChIP) assays.
FastScan™ ELISA Cell Extraction Enhancer Solution (50X) is used with FastScan™ ELISA Cell Extraction Buffer (5X) #69905 (not supplied) to prepare and dilute cell extracts for use in FastScan™ ELISA Kits.
This product is a concentrated fixation and permeabilization reagent designed for use with components of the FoxP3/Transcription Factor Fixation/Permeabilization Kit #43481. This reagent immobilizes cellular proteins and enables antibody binding of transcription factors localized within the nucleus, and is compatible with fluorescent detection by flow cytometry.Note: Precipitation may occur. The presence of precipitate does not affect the performance of the reagent.
This product is a diluent designed for use with components of the FoxP3/Transcription Factor Fixation/Permeabilization Kit #43481. Proper dilution of the FoxP3/Transcription Factor Fixation/Permeabilization Concentrate (4X) #44931 results in the reagent immobilizing cellular proteins and enabling antibody binding of transcription factors localized within the nucleus. This product is compatible with fluorescent detection by flow cytometry.Note: Precipitation may occur. The presence of precipitate does not affect the performance of the reagent.
Alexa Fluor® and many other anionic fluorescent dyes and proteins can bind nonspecifically with cationic cell and tissue constituents. By efficiently blocking these nonspecific electrostatic interactions, Image-iT® FX Signal Enhancer can dramatically improve the signal-to-noise ratio of immunolabeled cells and tissues. Image-iT® is a liquid that is applied directly to slides or coverslips containing fixed and permeabilized cell or tissue samples prior to staining with fluorescent probes.
Micrococcal nuclease is a relatively non-specific endo-exonuclease derived from Staphylococcus aureus. Active in the pH range of 7.0-10.0, this product digests double-stranded, single-stranded, circular, and linear nucleic acids.In the SimpleChIP® Enzymatic Chromatin IP kit assay, following cell lysis, the chromatin is fragmented by partial digestion with micrococcal nuclease to obtain chromatin fragments of 1 to 5 nucleosomes in size. Enzymatic digestion of chromatin is much milder than sonication and eliminates problems due to variability in sonication power and emulsification of chromatin during sonication, which can result in incomplete fragmentation of chromatin or loss of antibody epitopes due to protein denaturation and degradation.This product is offered to conveniently provide additional micrococcal nuclease when fragmenting chromatin with our SimpleChIP® (#9002, #9003) and SimpleChIP® Plus (#9004, #9005) Enzymatic Chromatin IP Kits. These kits provide all the reagents required for performing 6 chromatin preparations (or optimizations) and 30 chromatin immunoprecipitation (ChIP) assays, however there are instances where extra micrococcal nuclease is desired.
Premium quality normal goat serum for use as blocking reagent and antibody diluent for chromogenic and fluorescent immunohistochemical and immunocytochemical assays. Typically, normal serum from the same species as the secondary antibody is used in the blocking and diluent buffers.
Ultrapure Nuclease-free Water from Cell Signaling Technology is non-DEPC treated, endotoxin-tested, and contains no detectable endo- and exo-nucleases. Following stringent purification, it is 0.1 µm filtered into a polycarbonate bottle and autoclaved. It is suitable for molecular biology applications that require water devoid of nucleases.
This Phosphatase Inhibitor Cocktail is used to prevent dephosphorylation of phosphorylated proteins from active serine/threonine and tyrosine phosphatases present in whole cell extract. The 100X cocktail is a colorless to light yellow liquid.
Phenylmethanesulfonyl Fluoride (PMSF) is an inhibitor of serine proteases such as trypsin, chymotrypsin, thrombin, and papain. It is routinely added as a supplement to lysis buffers just prior to lysis, to prevent protease degradation. Cell Signaling Technology recommends adding PMSF at 1 mM to Cell Lysis Buffer (#9803) and RIPA Buffer (#9806). Molecular Formula: C7H7FO2S Molecular Weight: 174.19 CAS: 329-98-6
Ponceau S Staining Solution is supplied as ready to use. This product is recommended for rapid and reversible protein staining on nitrocellulose or PVDF membranes. This staining technique is often utilized to confirm protein electrotransfer in Western blotting assays prior to antibody-based detection.
When diluted in lysis buffer to a final concentration of 1X the Protease Inhibitor Cocktail prevents protein degradation by endogenous proteases present in whole cell extract. The 100XProtease Inhibitor Cocktail is a clear, colorless liquid.
When diluted in lysis buffer to a final concentration of 1X the Protease/Phosphatase Inhibitor Cocktail prevents protein degradation and dephosphorylation by endogenous proteases and phosphatases present in the whole cell extract. The 100X cocktail is a clear light yellow to light green liquid.
The Resazurin Cell Viability Kit is a fluorescent assay that detects cellular metabolic activity. The blue nonfluorescent resazurin reagent is reduced to highly fluorescent resorufin by dehydrogenase enzymes in metabolically active cells. This conversion only occurs in viable cells and thus, the amount of resorufin produced is proportional to the number of viable cells in the sample. The resorufin formed in the assay can be quantified by measuring the relative fluorescence units (RFU) using a fluorometer (Ex=530-570 nm, Em=590-620 nm).
SignalStain® Antibody Diluent is for use in immunohistochemical assays. It yields superior staining with a number of antibodies. This diluent can improve IHC signal and background; however, it is not compatible with all of Cell Signaling Technology's products that are recommended for use in immunohistochemical assays. Please consult the antibody datasheet to determine if SignalStain® Antibody Diluent is recommended for your product of interest.
SignalStain® Citrate Unmasking Solution (10X) is used for antigen unmasking of formalin fixed, paraffin-embedded tissue sections or cell pellets in immunohistochemical assays (IHC-P). Cell Signaling Technology recommends the optimal unmasking reagent for each IHC-P approved antibody. Please consult the primary antibody datasheet to determine if citrate is recommended for your specific product.