Benefits

β Amyloid ELISAs

β-Amyloid peptide (Aβ), a 40 to 43 amino acid peptide, is an early and critical feature of Alzheimer’s disease (AD). It is the major protein component of the protein deposits in neurofibrillary tangles, neuritic plaques, and neurophil threads in the cerebral cortex of AD and Down syndrome patients. The increased release of Aβ42/Aβ43 leads to the Aβnormal deposition of human Aβ and the associated neurotoxicity in the brains of affected individuals.^{1, 2}

Solid phase human Aβ40 and human Aβ42 ELISAs are designed for in vitro quantitative detection of human Aβ peptides in sera, cell culture supernatants and EDTA plasma and CSF. The assays recognize both natural and recombinant forms of human Aβ. The detection antibodies used in these kits are capAβle of selective differentiation between human Aβ40 and Aβ42/Aβ43. In both assays, an antibody specific for the NH2-terminus of human Aβ is coated onto the wells of a multiwell plate and binds human Aβ 1-40 or 1-42 present in samples or standards. A second antibody specific for the 1-40 or 1-42 sequence of human Aβ binds to the immobilized protein. After substrate addition, the reaction is visualized at 450 nm in the multiwell plate reader. The intensity of the color is directly proportional to the concentration of the human Aβ present in sample or standard. The assays are highly sensitive (detect human Aβ below 10 pg/mL), specific, fast (6.5 hours total time) and simple (offer precoated plates, all incubations proceed at room temperature and the reagents are supplied ready to use).

Features and Benefits
Size

• 96 wells

Sensitive

• Highly sensitive (below 10 pg/ml)

Specific

• Specifically measures Aβ40 or Aβ 42/43
• hAβ40 ELISA no cross-reactivity with hAβ42 or hAβ43 up to 10,000 pg/ml
• hAβ42 ELISA no cross-reactivity with hAβ40 or hAβ43 up to 10,000 pg/ml

Quantitative

• All results are quantitative
• Standard concentration curve is run in each assay

Simple

• Precoated plates eliminate the coating step
• All incubations proceed at room temperature
• Any commercially available microplate reader may be used

Fast

• Results in 6.5 hours

Economical

• Reagents may be used for multiple kit runs
• Cost per test is low

Performance Characteristics

• Precision
  • Very low intra-assay variations from 1.1% to 3.6%
  • Very low inter-assay variations from 1.7% to 3.7%
• Linearity of dilution – correlation coefficient for various dilutions was 0.99 for both assays
• Recovery – average recovery: sera 100%, EDTA plasma 92.8%, cerebrospinal fluid (CSF) 98%, cell culture supernatants 101-109%

Storage

• Store at 2 to 8°C

References

1. Sambamurti, K., et al., Neuromolecular Med., 1, 1-31 (2002).
2. Lorenzo, A., et al., Nat. Neurosci., 3, 460-464 (2000).
3. Vassar, R., et al., Science, 286, 735-741 (1999).

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Apoptosis Made Easy Kit

Features and Benefits

• High purity reagents
• CHX and ActD can also be used as tools to investigate the apoptosis inhibitors in these cells

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Flow Cytometry Kit for Apoptosis

Features and Benefits

• Stronger signal and improved detection as compared with FITC, biotin or digoxigenin-labeled Br-dUTP
• Better detection than fluorochrome (fluorescein or BODIPY)-conjugated deoxynucleotides
• Differentiation of non-fragmented DNA with propidium iodide/RNase A solution included in the kit
• Improved identification of non-apoptotic cells with fixed negative control cells included in the kit
• Sufficient for 60 cell suspensions

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Cathepsin Kits

Features and Benefits

• Fluorogenic substrates easily penetrate the cell membrane and the membranes of the internal cellular organelles
• athepsin intracellular localization identified and visualized by varying the duration and concentration of exposure to the fluorophore MR-cathepsin substrate
• Potent, cell-permeable and non-toxic fluorophore MagicRed™ reagent
• No need for cell lysis nor membrane permeabilization
• Kit could be run on fluorescence microscopes, fluorescence plate readers and flow cytometers
• Works on frozen cells
• Lysosomes and other intracellular organelles identified with acridine orange (AO) included in the kit
• Cell nuclei labeled with Hoechst stain included in the kit

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Cytochrome c Oxidase Assay Kit

Features and Benefits

• Detects mitochondrial outer membrane integrity
• Detects mitochondrial subcellular fractions
• Use with Sigma Mitochondrial Isolation Kit
• Suitable for 100 tests

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Cytochrome P450 Fluorescent Detection Kits

Features and Benefits

• Standard and substrates in solution ready to use
• Positive control provided
• Reagents for cell extraction provided
• Suitable for 50 assays

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Leukotriene and Prostanoid Enzyme Immunoassay (EIAs) Assays

Features and Benefits Specific

• Specifically measures for each target lipid
• Negligible cross-reactivity with 20-25 related eicosanoid compounds tested (from <0.1 to 2%)

Quantitative

• Standard curve is run in each assay

Simple

• Precoated plates eliminate the coating step
• All incubations proceed at room temperature
• Any commercially available multiwell plate reader may be used
• Reagents are color coded

Fast

• Results in 4 hours

Economical

• Reagents may be used for multiple kit runs

Performance Characteristics

• Low intra- and inter-assay variability
• Linear standard curve
• Recovery – ranges from 99-111%

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ELISA Kits

Solid phase sandwich Enzyme Linked-Immuno-Sorbent Assays (ELISA) employ an antibody (capture) specific for a protein (regardless of phosphorylation state) coated onto the wells of the multiwell plate. Samples, standards, control specimens and unknowns are incubated in the wells and antigen binds to the capture antibody. A second antibody specific for the protein in the assay (detection) is added and during the second one hour incubation, it binds to the immobilized protein captured during the first incubation. An anti-rabbit IgG-HRP completes the four-member sandwich. The reaction is visualized by the TMB substrate solution, which produces color, whose intensity is directly proportional to the concentration of the protein present in the original specimen. The reaction is stopped with a stop solution and read at 450 nm in the microtiter plate.

Features and Benefits Specific

• Specifically measures protein in questions with no crossreaction with other proteins

Sensitive

• Equal to or exceeds immunoblotting sensitivity

Convenient

• Kits contain all reagents needed for assay
• Precoated plates eliminate the coating step
• All incubations proceed at room temperature
• Any commercially available plate reader may be used

Accurate

• Control for total protein content with a pan protein ELISA

Quantitative

• Quantitate protein phosphorylation on a specific amino acid residue using a 96-well format

QC

• Lot-to-lot consistency of <10%

Validated

• Validated by peptide competition and mutant analysis ELISAs

Specificity

• Validated by peptide blocking and comparison with immunoblotting

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FAM-FLICA Caspase Assays

Fluorometric or flow cytometry detection of active cellular caspases using Fluorochrome Inhibitor of Caspase activity (FLICA).

Features and Benefits

• Easy, fast and more convenient
• Caspases are earlier indicators of apoptosis than Tunnel or Annexin V
• Potent, cell-permeable and non-toxic fluorochrome inhibitor
• Specific for selected caspase based on inhibitor specificity
• A direct measure of apoptosis expressed as the number of active caspase enzymes present in the cell
• No need for cell lysis nor membrane permeabilization
• Kit could be run on fluorescence microscopes, fluorescence plate readers and flow cytometers
• Works on majority of species because caspases are well conserved through the species

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Caspase Activity Assays

Fluorometric or flow cytometry detection of active cellular caspases using Fluorochrome Inhibitor of Caspase activity (FLICA).

Features and Benefits

• Easy, fast and more convenient
• Caspases are earlier indicators of apoptosis than Tunnel or Annexin V
• Potent, cell-permeable and non-toxic fluorochrome inhibitor
• Specific for selected caspase based on inhibitor specificity
• A direct measure of apoptosis expressed as the number of active caspase enzymes present in the cell
• No need for cell lysis nor membrane permeabilization
• Kit could be run on fluorescence microscopes, fluorescence plate readers and flow cytometers
• Works on majority of species because caspases are well conserved through the species

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Fluorometric Cell-Associated Nitric Oxide Synthase (NOS) Detection System

Sigma-RBI’s Fluorometric Cell-Associated Nitric Oxide Synthase Detection System is a simple and specific fluorometric assay for the measurement of free NO and NOS activity in living cells under physiological conditions. The need for radioisotope detection is replaced by the use of a cell-permeable diacetate derivative of the novel compound, 4,5-diaminofluorescein (DAF2-DA). The DAF2-DA penetrates cells rapidly and becomes hydrolyzed by an intracellular esterase to the parent DAF-2, which subsequently reacts with NO, produced by NOS, to form triazolofluorescein (DAF2-T). The fluorescent final product can then be quantified using an excitation filter at 492 nm and an emissions filter at 515 nm.

Fluorescence detection of NO production in control and in LPS/IFN-γ-induced Raw 264.7 murine macrophage cells cultured in 96-well plates. Fluorescence signals were obtained after an incubation at room temperature for 110 min. Results were calculated as the percentage of the maximal signals recorded with 1 mM arginine and 2.5 µM NADPH in induced cultures. DPI: Diphenyleneiodonium (iNOS selective inhibitor).

Features and Benefits

• Non-radioactive, cell based
• Uses cell-permeable fluorescein to detect intracellular NOS
• Kinetic quantitative reaction in a spectrofluorometer, linear for 2 hours at room temperature
• Specific - requires induction of iNOS activity and inhibition of fluoresence generation by NOS inhibitors
• Convertible to high throughput screening for drug discovery efforts

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Hydrogen Peroxide (H₂O₂) Colorimetric Assay

Measurement of H₂O₂ helps to determine how oxidative stress modulates various intracellular pathways. The H₂O₂ kit is designed to measure low concentrations of H₂O₂ in biological matrices. A color reagent produces a purple color proportional to the concentration of H₂O₂ in the samples or standards. The mechanism of the color reaction probably involves coordinated iron reacting with H2O2 and the dye molecule. The color reaction is read at 540-570 nm in the microplate spectrophotometer. Plot absorbance versus standard H₂O₂ concentrations to quantitate H₂O₂ in the samples.

Samples

• A wide range of samples may be used and diluted with 50 mM phosphate, pH 6.0 or cell culture media

Features and Benefits
Simple

• No sample extraction or lysis required
• One step procedure with one reagent

Sensitive

• Detects H₂O₂ at 51.25 ng/ml when diluted in buffer

Quantitative

• All results are quantitative
• Standard concentration curves are run in each assay

Very Fast

• Results available within 30 minutes

Precision

• Intra assay variations 3-10%
• Inter-assay variations 1.7-5.7%

Linearity

• A dilution of hydrogen peroxide was run multiple times in same assay
• The obtained line has a slope of 0.9342 with CV of 0.9983
• Plasma, urine and serum samples diluted 1:64 gave 90.6-105.6% recovery

Recovery

• Plasma, urine and serum samples spiked with H₂O₂ and diluted 1:64 gave >90% recovery

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Kinase Assays –p38, JNK, MEK

Assays measure kinase phosphorylation activity, activation and inhibition by immunoblot detection. The endogenous kinase of interest is immunoprecipitated using the kinase specific antibody and EZview Red Protein A. The immunoprecipitated kinase phosphorylates a specific substrate. The kinase activity is then detected by an immunoblot analysis of the phosphorylated substrate using phosphospecific antibodies.

Features and Benefits

• A non-radioactive sensitive assay
• Optional radioactive protocol
• Contains all required reagents for the immunoprecipitation and phosphorylation reactions and also a detection antibody
• Easy and convenient procedure
• Friendly to use due to its components
  • The EZview Red Affinity Gel: allows a better visualization of the precipitated fraction
  • HRP conjugated detection antibody (JNK and p38 MAPK Activity Assay Kits component): no requirement for a secondary antibody, thus, reducing background activity
• p38 MAPK and MEK Activity Assay Kits contain a specific inhibitor for further verification of the kinase specificity
• Procedure is relatively short
• Reproducible results
• Useful for
  • Studying kinases pathways
  • Studying kinases, activators and inhibitors
  • Studying and identifying extracellular stress stimuli that are converted into specific cellular responses through the activation of the specific kinase signaling pathway

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Mitochondria Isolation Kit

The Mitochondria isolation kit enables the fast and easy isolation of an enriched mitochondrial fraction from animal tissues as well as the testing of the electrochemical proton gradient of the inner mitochondria-mediated apoptosis.

Features and Benefits

• Measures integrity of inner mitochondrial membrane (using fluorescent carbocyanine dye JC-1 supplied in the kit)
• Useful for isolating mitochondrial proteins for proteasome studies
• Can use Sigma’RBI’s Cytochrome c Oxidase Assay kit to measure outer membrane activity
• Suitable for 50 assays

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Phosphospecific ELISAs

Phosphospecific ELISAs offer a sensitive, quantitative and economical alternative to traditional immunoblotting or functional assays.

The format is a solid phase sandwich Enzyme-Linked Immunosorbent Assay (ELISA). A capture antibody specific for a protein (regardless of phosphorylation state) has been coated onto the wells of a multiwell plate. Antigen binds to the capture antibody. A detection antibody specific for the phosphorylated or nonphosphorylated protein is added and binds to the immobilized protein. An anti-rabbit IgG-HRP completes the four member sandwich. The reaction is visualized by the tetramethylbenzidene (TMB) substrate and the intensity of the color is directly proportional to the concentration of protein present in the original sample. The reaction is read at 340 nm in a multiwell plate reader.

Cell extracts are the samples of choice, because the proteins involved in the phosphorylation are present in intracellular compartments. Researchers may use the cell extraction procedure provided in the technical bulletin or utilize the Sigma Mammalian Cell Lysis Kit.

Features and Benefits
Sensitive

• picograms/ml quantities for non-phosphospecific ELISAs
• below 1.0 unit/ml for phoshospecific kits
• 2 to 10 times more sensitive than traditional immunoblots

Sensitive

• Highly specific for target protein and phosphorylation site
• Validated against immunoblotting performed with the same antigen and antibodies
• Validated by peptide competition

Quantitative

• All results are quantitative
• Standard concentration curve is run in each assay

Simple

• Precoated plates eliminate the coating step
• All incubations at room temperature
• Small sample size – only 10 µl (diluted to a total volume of 100 µl)
• Use any multiwell reader available on the market
• No additional equipment required

Stable

• Entire kit is stable in the refrigerator for 12 months
• Components are all stored at the same temperature
• Liquid stable chromogen and stop reagents
• Two vials of standard extend the kit life span

Fast

• Results in 4 hours

Economical

• Reagents may be used for multiple kit runs

Performance Characteristics

• Intra and Inter-assay variations
• Linearity of dilution
• Recovery – ranges from 93 to 97%
• Each phosphospecific kit is normalized against non-phosphospecific ELISA

Sample Data Using Sigma-RBI Phosphospecific ELISAs

Detection of p38 MAPK [pThr¹⁸⁰/pTyr¹⁸²] by ELISA vs. Immunoblotting. The sensitivity of this ELISA was compared to immunoblotting using known quantities of p38 MAPK [pThr180/pTyr182]. The data presented show that the sensitivity of the ELISA is approximately 10X greater than that of immunoblotting. The bands shown in the immunoblotting data were developed using rabbit anti-p38 MAPK [pThr¹⁸⁰/pTyr¹⁸²], an alkaline phosphatase conjugated anti-rabbit IgG, followed by chemiluminescent substrate and autoradiography.

Recombinant p38 MAPK was phosphorylated using MKK6 enzyme in vitro. Non-phosphorylated p38 MAPK was used as control. The phosphorylated and non-phosphorylated p38 MAPK were analyzed with phospho-p38 MAPK [pThr¹⁸⁰/pTyr¹⁸²] ELISA and p38 MAPK ELISA. This phospho-p38 MAPK (pThr¹⁸⁰/pTyr¹⁸²) ELISA kit is specific for measurement of phosphorylated p38 MAPK at threonine 180 and tyrosine 182. The kit does not detect non-phosphorylated p38 MAPK protein.

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Glutathione Assay Kit: For the measurement of total glutathione in biological samples

Reduced glutathione (GSH), a tripeptide (γ-glutamyl-cysteinylglycine), is involved in many biological processes such as detoxification of xenobiotics, removal of hydroperoxides, and maintenance of the oxidation state of protein sulfhydryls. It is the key antioxidant present in animal tissues.

The kit provides all reagents for the simple and quick assay of the level of total glutathione (oxidized glutathione and GSH) in a cell, tissue extracts, red blood cells or plasma.

The biological samples are first deproteinized using 5% sulfosalicylic acid solution. The total glutathione content of the sample is then assayed using a kinetic assay in which catalytic amounts of glutathione cause a continuous reduction of 5,5’-dithiobis-(2-nitrobenzoic acid (DTNB) to TNB. The oxidized glutathione formed is recycled by glutathione reductase and NADPH. The product, TNB, is assayed colorimetrically at 412 nm.

Assaying in a 96 well plate maximizes the number of samples that can be tested.

Features and Benefits

• picograms/ml quantities for non-phosphospecific ELISAs
• below 1.0 unit/ml for phoshospecific kits
• 2 to 10 times more sensitive than traditional immunoblots

Sensitive

• A quick and simple method
• The kit provides all the reagents required for a colorimetric assay of total glutathione (oxidized + GSH)
• The kit includes a deproteination reagent for biological samples

Standard curve of reduced glutathione as measured in the kinetic colorimetric assay:

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Glutathione S-Transferase (GST) Assay Kit: For the measurement of GST in biological samples

Glutathione S-transferases (GSTs) are a group of enzymes important in the detoxication of many xenobiotics in mammals. The enzymes protect cells against toxicants by conjugating the thiol group of the glutathione to electrophilic xenobiotics, and thereby defend cells against the mutagenic, carcinogenic, and toxic effects of the compounds. GST activity is present in plants, insects, yeast, bacteria, and in most mammalian tissues, especially in the liver, which plays a key role in detoxification.

Glutathione S-Transferase (GST) Assay Kit assays total GST activity by measuring the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with reduced glutathione. Product formation (GS-DNB conjugate) is detected at 340 nm, which makes possible a spectrophotometric assay for GST activity. The rate of increase of absorption is directly proportional to the GST activity in the sample.

Features and Benefits

• All the reagents required for a fast and easy measurement of total GST activity
• A GST positive control
• Used to measure GST activity in cell and bacterial lysates, tissue homogenates and in plasma and erythrocyte lysates
• Sufficient for 100 assays in 1 ml cuvettes or 500 reactions in 96 well plates

Measurement of Glutathione-S-Transferase (GST) activity:

The activity of GST enzyme was measured at different concentrations in a 1 ml cuvette using the GST Assay Kit. The graph demonstrates a linear ratio between the amount of enzyme (over the range ~0.05-0.5 µg) and the enzyme activity, as measured by the increase in OD³⁴⁰ units per min.

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Casein Kinase Kits

The three casein kinase kits provide an easy method for measuring the activity of the different casein kinases by an in vitro phosphorylation of casein substrates. The kits can be used with cell lysates, tissue homogenates, column fractions, or the purified enzyme.

Casein kinases (CK) are highly conserved serine/threonine kinases, ubiquitous in eukaryotic organisms, and defined by their substrate preference for casein. They are insensitive to cyclic nucleotides, calcium, and phospholipids. Casein kinases are grouped into two main families, type I and type II, according to their structure, specificity and response to effectors.

Features and Benefits

• Easy method for measuring the activity of the different casein kinases by in vitro phosphorylation of substrate
• Includes specific inhibitors to allow the distinction between the CK activities
• Can be used for CK activity measurement in cell lysates, tissue homogenates, column fractions or a purified enzyme

Casein Kinase Assay Kit

The Casein Kinase Assay Kit (general) measures the activity of the different casein kinases by an in vitro phosphorylation of α-casein. The a-casein substrate is phosphorylated by both CKI and CKII. The kits includes CKI and CKII specific inhibitors to distinguish between CKI and CKII activities in the biological sample.

Casein Kinase I Assay Kit

The Casein Kinase I (CKI) Assay Kit measures the activity of Casein Kinase I by an in vitro phosphorylation of a Casein Kinase I phosphopeptide substrate (a specific CKI substrate). In addition, the kit contains a CKI specific inhibitor (IC261) to ensure the specificity of the CK activity measured and a purified CKId enzyme to serve as a positive control.

Casein Kinase II Assay Kit

The Casein Kinase II Assay Kit measures the CKII enzyme activity by an in vitro phosphorylation of the Casein Kinase II Substrate. This substrate is a peptide that is selective for CKII with not activity with CKI. The kit also contains a CKI specific inhibitor to ensure the specificity of the CK activity and in addition, it also includes purified CKII for use as a positive control.

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DHFR Assay Kit: For the detection of Dihydrofolate Reductase (DHFR) activity and for screening of DHFR inhibitors

DHFR is an enzyme catalyzing a reaction essential for the biosynthesis of nucleotidic bases of DNA. Blockade of the DHFR enzyme causes cell death as a result of inhibition of DNA synthesis. For this reason, DHFR is considered a target for the development of anti-tumor drugs.

The assay is based on the ability of DHFR to catalyze the reversible NADPH-dependent reduction of dihydrofolic acid to tetrahydrofolic acid. The reaction progress is followed by monitoring the decrease in absorbance at 340 nm.

Features and Benefits

• Contains all the reagents required for a colorimetric assay of DHFR activity in cell lysates, tissue homogenates, or column fractions of purified enzyme
• Includes a purified enzyme to be used as a positive control and for screening of DHFR inhibitors
• Includes methotrexate (MTX), a prokaryotic and eukaryotic DHFR specific inhibitor, which exhibits anti-tumor activities
• Tested using A431, NIH 3T3 and CHO cell lines, rat liver, kidney, brain, and skeletal muscle tissue extracts and recombinant DHFR
• Sufficient reagents are supplied for 50-100 1ml tests

Activity of recombinant Dihydrofolate Reductase (DHFR) in the presence of the inhibitor methotrexate (MTX). Specific activity (Unit/mg protein) of recombinant DHFR was measured in the absence or in the presence of different concentrations of MTX. 50% inhibition of the enzyme is observed at a concentration of 0.025 µM MTX.

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New Retinoblastoma Phospho-Specific ELISAs and Antibodies

Features and Benefits

• Very low intra-assay variability from 2.84% to 7.58%
• Very low inter-assay variability from 5.7% to 9.21%
• Linearity of dilution – correlation coefficient for various dilutions was 0.99 for both assays
• Recover – average recovery: 92.7% and 10% respectively
• Suitable for 96 wells

Human Rb ELISA is designed for in vitro quantitative detection of non-phosphorylated, phosphorylated and dually phosphorylated human Rb in cell lysates. A monoclonal capture antibody specific for Rb protein (regardless of phosphorylation state) has been coated onto a 96 multiwell plate. Rb antigen in samples and standards binds to the capture antibody. A detection antibody, specific for the phosphorylated or non-phosphorylated human Rb, is added and binds to the immobilized Rb protein. A streptavidin-HRP or anti-rabbit IgG-HRP completes the four-membrane sandwich. The reaction is visualized by a TMB substrate and the intensity of the color is directly proportional to the concentration of Rb present in the original sample. The reaction is read at 450 nm in a multiwell plate reader. The assays detect both native and recombinant human Rb and phospho-Rb [pSer²⁴⁹/pThr²⁵²] and pThr⁸²¹] proteins. The assays are highly sensitive (<0.1 ng/mL and <0.8 units/mL, respectively), specific fast (4 hours total time) and simple (offer precoated plates, all incubations proceed at room temperature and the reagents are supplied ready to use).

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