Phosphoprotein Extraction & Detection Products
A frequently used activation mechanism in signal transduction systems is stimulation of protein kinases.
When a protein kinase is stimulated to phosphorylate another protein, this action initiates an intracellular biological response to the extracellular signal. Often a protein phosphatase dephosphorylates the phosphoproteins to terminate the biological response. Protein phosphorylation/dephosphorylation systems consist of at least three components: (a) phosphoproteins, which alter their properties during phosphorylation/dephosphorylation; (b) protein kinases, which transfer a phosphate group from a nucleotide triphosphate (often ATP) to specific serine, threonine, or tyrosine residues on phosphoproteins; and (c) protein phosphatases, which dephosphorylate phosphoproteins to return the particular protein phosphorylation system to its initial state.
On this page, we list products designed for the convenient and rapid isolation of phosphoproteins along with antibodies to phosphoserine, phosphotyrosine, phosphothreonine, and related reagents.
PhosphoSafe Extraction Buffer extracts cytosolic proteins from mammalian and insect cells while preserving their phosphorylation state. The reagent, an enhancement on our CytoBuster Protein Extraction Reagent, includes four phosphatase inhibitors: sodium fluoride, sodium orthovanadate, b-glycerophosphate, and sodium pyrophosphate. This convenient, ready-to-use formulation, allows for the rapid extraction of phosphoproteins in a formulation that is compatible with Western blot analysis, immunoprecipitation, and assays for cytoplasmic kinase activity.
Figure 1. Western blot analysis of phosphorylated MLC2 after extraction from L6 myoblasts with CytoBuster reagent or PhosphoSafe Extraction Buffer
Figure 1 demonstrates that PhosphoSafe buffer preserves the phosphorylation state of proteins. Here, we used an antibody against myosin light chain 2 (anti-phospho-MLC2) that specifically recognizes the combination of phosphorylated threonine and serine at amino acid positions 18 and 19, respectively, in a Western blot analysis of the protein extracts prepared from L6 myoblasts using Cytobuster and Phosphosafe. Protein extracts prepared with PhosphoSafe buffer produced a more intense detection signal than did extracts prepared without it.
Figure 2. Kinase assay results for PKA and PKC for CHO-K1 cell extracts prepared with CytoBuster reagent or PhosphoSafe Extraction Buffer.
Here, we incubated biotinylated peptides corresponding to the PKA phosphorylation site of HNF-6 and the pseudosubstrate region of PKC with increasing amounts of extract (0.6 mg/ml) in the presence of γ-32P ATP using the Protein Kinase Assay Kit, Universal. Phosphate transfer mediated by Protein Kinase A (PKA; panel A) and Protein Kinase C (PKC; panel B) was detected by scintillation counting. Protein concentration was determined using the BCA Protein Assay Kit.
Protein phosphorylation-dephosphorylation is one of the major signaling mechanisms for modulating the functional properties of proteins involved in gene expression, cell adhesion, cell cycling, cell proliferation, and differentiation. Phosphorylation occurs on specific serine, threonine, or tyrosine residues by the action of protein kinases, which catalyze the transfer of a phosphoryl group from a high-energy compound (e.g., ATP, GTP) to a nucleophilic acceptor group on the amino acid side-chain(s) of proteins. The signaling mechanism relies on two reversible reactions that include phosphorylation by protein kinases and water-driven hydrolysis of phosphoester bonds by protein phosphatases. Choose from our set of nearly 200 phosphospecific antibodies that discriminate between the phosphorylated and non-phosphorylated states of your protein of interest.
The ProteoEnrich™ ATP-Binders™ Kit allows group separation of protein kinases and other ATP binding proteins, yielding partially purified cell extracts enriched in protein kinases that retain enzymatic activity. Based on an affinity resin containing immobilized ATP, the method is compatible with 2D electrophoresis, SDS-PAGE/tandem mass spectrometry, Western blot analysis, or activity assays. The unique affinity resin contains ATP covalently linked through the g-phosphate, presenting an ideal configuration for recognition by the conserved ATP binding pocket of kinases.
Antibodies and Detection Tools for Phosphoamino Acids
|Cat. No.||Product Name||Product Description|
|Phosphoprotein Enrichment Kit||This kit provides a quick and easy method for the enrichment of phosphoproteins from tissues or cell culture for immunoblotting. It contains columns, wash buffer, elution buffer, lysis buffer, and SDS-PAGE sample buffer sufficient to process up 25 g of tissue or 109 cells.|
|525282||Phosphoserine Detection Kit||A set of four different monoclonal antibodies specific for serine phosphorylation sites (clones 4A3, 4A9, 1C8, and 16B4). Reacts with Arabidopsis, bacteria, chicken, human, maize, mouse, rat, tobacco, Xenopus, yeast, and zebrafish.|
|525283||Anti-Phosphoserine (4A3) (Mouse)||These antibodies recognizes a broad range of serine-phosphorylated proteins in crude cell extracts, preferring positively charged amino acids directly flanking the phosphoserine. Purified from serum-free cell culture supernatant by thiophilic adsorption and size exclusion chromatography. React with human, mouse, rat, chicken, Xenopus, zebrafish, tobacco, maize, Arabidopsis, yeast, and bacterial proteins containing phosphoserine. Isotype: IgM.|
|525284||Anti-Phosphoserine (4A9) (Mouse)|
|525281||Anti-Phosphoserine (1C8) (Mouse)|
|525280||Anti-Phosphoserine (16B4) (Mouse)||Recognizes either -Ser-Pro- or -Ser-Lys- sequences that have been phosphorylated on the serine residue. Reacts with human, mouse, rat, chicken, Xenopus, zebrafish, tobacco, maize, Arabidopsis, yeast, and bacterial proteins containing phosphoserine. Isotype: IgM.|
|525288||Phosphothreonine Detection Kit||A set of three different monoclonal antibodies specific for threonine phosphorylation sites (clones 14B3, 1E11, and 4D11). The phosphorylation patterns in a given cell extract may differ when probed with individual antibodies due to sequence specificity. Recognition depends on phosphorylation and the surrounding amino acid motif. Reacts with threonine–phosphorylated proteins in Arabidopsis, bacteria, chicken, human, maize, mouse, rat, tobacco, Xenopus, yeast, and zebrafish. Supplied with a positive control lysate.|
|525286||Anti-Phosphothreonine (14B3) (Mouse)||Monoclonal antibody recognizes phosphorylated threonine residues. Purified from serum-free cell culture supernatant by thiophilic adsorption and size exclusion chromatography. Recognizes phosphorylated threonine in proteins from human, mouse, rat, chicken, Xenopus, zebrafish, tobacco, maize, Arabidopsis, yeast, and bacterial cells. Isotype: IgG1.|
|525287||Anti-Phosphothreonine (1E11) (Mouse)||Recognizes a broad range of threonine-phosphorylated proteins in crude cell extracts, serving as a valuable tool for non-radioactive phosphothreonine detection. Purified from serum-free cell culture supernatant by thiophilic adsorption and size exclusion chromatography. Reacts with human, mouse, rat, chicken, Xenopus, zebrafish, tobacco, maize, Arabidopsis, yeast, and bacterial proteins containing phosphothreonine. Isotype: IgG1.|
|525322||Phosphotyrosine Detection Kit||A set of four different monoclonal antibodies specific for tyrosine phosphorylation sites (see more about these four antibodies). Reacts with tyrosine–phosphorylated proteins in chicken, human, mouse, rat, Xenopus, yeast, and zebrafish.|
|525324||Phosphotyrosine Molecular Weight Markers||Lyophilized solid. Contains seven phosphotyrosine-modified proteins ranging in molecular weight from 21 to 114 kDa. Markers are b-galactosidase, 114 kDa; phosphorylase A, 94 kDa; conalbumin, 78 kDa; BSA, 67 kDa; ovalbumin, 47 kDa; carbonic anhydrase, 30 kDa; soybean trypsin inhibitor, 21 kDa. Each kit is sufficient to perform 50–100 minigels. Also useful as a standard for the Phosphotyrosine Detection Kit.|
|PT01L||Anti-Phosphotyrosine (Ab-1) (Mouse)||Reacts with phosphotyrosine-containing proteins such as the receptors for EGF and PDGF. Does not cross-react with phosphoserine, phosphothreonine, or phosphohistidine nor with a variety of other phosphorylated molecules including ribose phosphate, IMP, AMP, or ATP. For paraffin sections, pretreat with saponin. Isotype: IgG1.|
|PT02||Anti-Phosphotyrosine (Ab-1) (Mouse) Agarose Conjugate||Agarose conjugate of Ab-1.|
|525293||Anti-Phosphotyrosine (2C8) (Mouse)||Recognizes a broad spectrum of tyrosine-phosphorylated proteins in crude cell extracts. Reacts with phosphotyrosine in human, mouse, rat, chicken, Xenopus, yeast, and zebrafish lysates. Not suitable for use with bacterial lysates. Lyophilized solid, purified. Isotype: IgG1.|
|525295||Anti-Phosphotyrosine (PY20) (Mouse)||These antibodies are specific for tyrosine–phosphorylated proteins in the native and denatured states. Binding is inhibited by phosphotyrosine; does not react with threonine– or serine–phosphorylated residues on proteins. Liquid. Isotype: IgG2b.|
|525300||Anti-Phosphotyrosine (PY20) (Mouse) Agarose Conjugate|
|525310||Anti-Phosphotyrosine (PY20) (Mouse) Biotin Conjugate|
|525320||Anti-Phosphotyrosine (PY20) (Mouse) Peroxidase Conjugate|
|525290||Anti-Phosphotyrosine (Rabbit)||Specific for phosphotyrosine-containing proteins. Liquid, affinity purified. Isotype: IgG.|
|Cat. No.||Product Name||Product Description|
|69266||Goat Anti-Mouse IgG AP Conjugate (H + L)||Optimized for maximal signal:noise in Western blotting and plaque/colony screening applications. This conjugate is prepared from affinity-purified anti-IgG with the specificities indicated. For ELISA applications, the optimal working dilution is higher than for blots (i.e., up to 1:50,000).|
|71045||Goat Anti-Mouse IgG HRP Conjugate (H + L)||Optimized for maximal signal:noise in Western blotting and plaque/colony screening applications. This conjugate is prepared from affinity-purified anti-IgG with the specificities indicated. For ELISA applications, the optimal working dilution is higher than for blots (i.e., up to 1:50,000).|
|69265||Goat Anti-Rabbit IgG AP Conjugate (Fc specific)||Optimized for maximal signal:noise in Western blotting and plaque/colony screening applications. This conjugate is prepared from affinity-purified anti-IgG with the specificities indicated. For ELISA applications, the optimal working dilution is higher than for blots (i.e., up to 1:50,000).|
|70955||5% Alkali-soluble Casein||Alkali-soluble casein is often recommended as a blocking reagent for use on Western blots.|
|69264||AP Detection Reagent Kit||The AP Detection Reagent Kit includes standardized solutions of 3-bromo-4-chloro-5-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT), plus 20X AP Buffer, for sensitive chromogenic detection of alkaline phosphatase conjugates. With the NBT/BCIP system, positive bands turn a deep blue-violet color that resists fading.|
|69086||CDP-Star® AP Substrate||The CDP-Star® AP Substrate enables subnanogram sensitivity in a convenient ready-to-use format, and includes Nitro-Block II™ signal enhancer for increased signal-to-noise ratios with standard nitrocellulose membranes.|
|69059||SuperSignal® HRP Substrate||The SuperSignal® HRP Substrate enables subnanogram sensitivity in a convenient, ready-to-use format.|