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LightShift™ Chemiluminescent RNA EMSA Kit (Thermo Scientific™)

The Thermo Scientific LightShift Chemiluminescent RNA EMSA Kit provides a non-radioactive solution for studying RNA-protein interactions using an electrophoretic mobility-shift assay (EMSA).

Features of the LightShift Chemiluminescent RNA EMSA Kit:

Sensitive—chemiluminescent detection comparable to radioactive detection
Time-saving—develop X-ray films after 1- to 5-minute exposures, versus 16 hour exposures needed with radioactive systems
Flexible—compatible with RNA probes biotinylated by different methods
Easy to use—complete kit includes optimized reagents for binding reactions and RNA probe detection
Non-radioactive—eliminate waste concerns from radioactive RNA probes

The RNA EMSA Kit uses biotinylated RNA probes and a chemiluminescent substrate system to achieve fast and safe detection of RNA-protein complexes with sensitivity equivalent to traditional 32P-isotopic methods. The complete kit comes with all reagents required to set up and optimize protein-RNA binding conditions, a positive control for protein-RNA interactions and reagents for chemiluminescent detection of the nucleic acid interaction.

About the LightShift Chemiluminescent RNA EMSA Kit
The LightShift Chemiluminescent RNA EMSA Kit is an in vitro technique for detection of protein-RNA interactions through changes in gel electrophoresis migration patterns similar to the popular DNA gel shift assay. In a RNA EMSA, a labeled RNA probe is incubated with a protein sample to initiate binding. Once a complex is formed, the sample is separated via non-denaturing polyacrylamide gel electrophoresis. Because RNA-protein complexes migrate more slowly than free RNA probes, the resulting difference in migration distance can be visualized with the RNA gel shift assay. Specificity of RNA-protein interactions are validated through binding competition with excess unlabeled RNA that decrease the signal of the specific interactions. Generally, mutated or irrelevant RNA probes are not expected to compete for specific interactions and should not reduce the intensity of specific band shifts when detected in the EMSA. The complete LightShift Chemiluminescent RNA EMSA Kit includes all reagents required to set up and optimize an RNA gel shift assay, including a positive control for RNA-protein complex formation.

The LightShift Chemiluminescent RNA EMSA Kit uses biotinylated RNA probes, streptavidin-HRP and chemiluminescent detection to provide sensitivity similar to using radioactive RNA probes but with faster detection. Labeled RNA probes can be purchased commercially or generated through either run-off in vitro transcription reactions with biotinylated nucleotides or through enzymatic ligation of biotin tags to the 3' terminus of an RNA strand using the Thermo Scientific Pierce RNA 3' End Biotinylation Kit. The LightShift Chemiluminescent RNA EMSA Kit is effective for RNA probes biotinylated by any of these three methods; however RNA secondary structure may be affected by internal incorporation of biotinylated nucleotides during run-off in vitro transcription RNA probe synthesis. Therefore, for certain interactions, custom synthesized RNA probes or 3' end biotinylated probes may be required for proper protein-RNA interactions to occur.

About the LightShift Chemiluminescent RNA EMSA Kit Positive Control
The positive control included with the LightShift Chemiluminescent RNA EMSA Kit is the iron responsive element (IRE) RNA probe. IRE binding reactions are set up and detected in parallel with the other experimental samples. Under iron-starved conditions, the iron responsive protein (IRP) remains bound to the IRE RNA present in the cell, effectively suppressing translation of ferritin (an iron storage protein) and then transferrin iron receptor. Under iron-rich conditions, IRE binding activity is lost, and ferritin and transferrin are translated. This system is ubiquitous and yields a robust band shift. Incubating the positive control reaction with a 200-fold molar excess of unlabeled IRE RNA will reduce the specific IRE band-shift signal by approximately 70%, while a similar fold excess of an unrelated RNA probe will not significantly reduce the IRE band shift. These controls can be used in each RNA gel shift experiment to validate proper setup, electrophoresis, transfer and detection of the protein-RNA complex formation.

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tRNA for LightShift™ Chemiluminescent RNA EMSA Kit

LightShift™ Poly (dI-dC) (Thermo Scientific™)

Thermo Scientific LightShift Poly(dI-dC) can be used as competitor for nonspecific DNA binding proteins.

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

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LightShift™ Chemiluminescent EMSA Kit
LightShift™ EMSA Optimization and Control Kit

tRNA for LightShift™ Chemiluminescent RNA EMSA Kit (Thermo Scientific™)

tRNA, formulation: Transfer RNA at 10 mg/mL in 10mM HEPES.
Sufficient For: 100 binding reactions

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LightShift™ Chemiluminescent RNA EMSA Kit

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
LightShift™ Poly (dI-dC)

SphingoStrips™ Membranes (Invitrogen™)

SpingoStrips™ membranes are designed for the identification of proteins possessing phosphoinositide or sphingolipid recognition domains and for analysis of their lipid-binding specificities.

Pierce™ Peroxide Assay Reagent C (Thermo Scientific™)

Thermo Scientific Pierce Peroxide Assay Reagent C is a formulation consisting of 125µM xylenol orange in methanol with BHT. This formulation is one of the component of Quantitative Peroxide Assay Kit used for detecting and measuring hydrogen peroxide levels based on the oxidation of ferrous to ferric ion in the presence of xylenol orange, present in the biological and other liquid samples.

Features of Peroxide Assay Reagent B:

Convenient—no proprietary reagents involved and remove the variability, difficulty and added expense of formulating the reagents yourself
Lipid-compatible formulations— the lipid-compatible formulation provides the best results with lipid-containing samples without first extracting the lipids;however, because this kit does not contain sorbitol, the assay is less sensitive.

Applications of Pierce Quantitative Peroxide Assay Kits:
• 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 these assay kits, hydroperoxides convert the Fe2+ to Fe3+ at acidic pH. With the aqueous-compatible formulation, peroxide first reacts with sorbitol, converting it to a peroxyl radical, which in turn initiates Fe2+ oxidation to Fe3+. In the 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 know extinction coefficient of the XO-Fe complex or by reference to a standard curve prepared with hydrogen peroxide solution.Most proteins do not interfere with these assays, although some metal chelators may require use of a blank (i.e., control) to mitigate these effects.


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Pierce™ Quantitative Peroxide Assay Kit (Aqueous)
Pierce™ Quantitative Peroxide Assay Kit (Lipid)
Pierce™ Peroxide Assay Reagent A
Pierce™ Peroxide Assay Reagent B

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™ Quantitative Peroxide Assay Kit (Lipid)
Pierce™ Peroxide Assay Reagent A
Pierce™ Peroxide Assay Reagent B
Pierce™ Peroxide Assay Reagent C

Glycoprotein Standards Set for Pierce™ Glycoprotein Carbohydrate Estimation Kit (Thermo Scientific™)

The Thermo Scientific Pierce Glycoprotein Standards Set includes six purified standards (two negative and four positive controls) intended for use with any glycoprotein carbohydrate determination protocol.

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Pierce™ Glycoprotein Carbohydrate Estimation Kit

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

The Thermo Scientific Pro-Detect Rapid His Competitive Assay Kit is a single-use, dipstick lateral flow kit for quick, easy detection of poly-histidine (His)-tagged proteins in protein expression and purification systems. This lateral flow assay uses membrane-based strips coated with dye-bound conjugated antibodies specific to His tag to provide a qualitative detection within ten minutes. This assay can be used to detect His-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™ Chromogenic Endotoxin Quant Kit (Thermo Scientific™)

The Thermo Scientific Pierce Chromogenic Endotoxin Quant Kit is a highly sensitive endpoint assay that accurately measures and detects endotoxin (lipopolysaccharide) in a protein, peptide, nucleic acid, or antibody sample using the amebocyte lysate assay. The kit enables detection within two linear sensitivity ranges of 0.01–0.1 EU/mL and 0.1–1.0 EU/mL.

Features and benefits of the Pierce Chromogenic Endotoxin Quant Kit include:
Highly sensitive with a broad range—detect as little as 0.01 EU/mL to 1 EU/mL
Specific—no interference from ß-glucans and suitable for wide range of samples, including protein, vaccine, plasmid, DNA, RNA
Fast—perform assay in as little as 20 minutes
End-point chromogenic assay—measure with a standard spectrophotometer or plate reader at 405–410 nm

The Pierce Chromogenic Endotoxin Quant Kit is an end-point chromogenic endotoxin detection assay based on the amebocyte lysate method, which measures endotoxin through the interaction of the endotoxin with the proenzyme Factor C found in circulating amebocytes of the horseshoe crab. The proteolytic activity of this proenzyme is activated in the presence of lipopolysaccharides (endotoxins) derived from the outer cell membrane of gram-negative bacteria such as E.coli. Endotoxin levels are determined by measuring the activity of Factor C in the presence of a synthetic peptide substrate that releases p-nitroaniline (pNA) after proteolysis, producing a yellow color that can be measured at an absorbance of 405 nm.

Endotoxin levels in the samples are accurately determined using the included endotoxin standard of known concentration that is derived from E.coli strain O111: B4. Determining endotoxin levels is important to assess the efficiency of endotoxin removal methods and prevent endotoxic shock, inflammation, and/or sepsis in tissue culture cells and animals injected with endotoxin-contaminated proteins.

Applications: quantitation of endotoxin levels in a protein, peptide, antibody, or nucleic acid samples

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Pierce High Capacity Endotoxin Removal Resin
Pierce High Capacity Endotoxin Removal Spin Columns

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

The Thermo Scientific Pro-Detect Rapid Myc Competitive Assay Kit is a single-use, dipstick lateral flow kit for quick, easy detection of Myc (sequence: EQKLISEEDL)-tagged proteins in protein expression and purification systems. This lateral flow assay uses membrane-based strips coated with dye-bound conjugated antibodies specific to Myc tag to provide a qualitative detection within ten minutes. This assay can be used to detect Myc-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.

PIP Strips™ Membranes (Invitrogen™)

PIP Strips™ membranes are designed for the identification of proteins possessing phosphoinositide recognition domains and for analysis of their lipid-binding specificities. PIP Strips™ membranes facilitate the analysis of phosphoinositide protein interactions by protein-lipid overlay assays. Proteins may be detected using standard western blot procedures in conjunction with high-performance alkaline phosphatase- and horseradish peroxidase (HRP)-mediated signal generation systems.

Electrophoretic Mobility-Shift Assay (EMSA) Kit, with SYBR™ Green & SYPRO™ Ruby EMSA stains (Invitrogen™)

This fluorescence-based Electrophoretic Mobility Shift Assay (EMSA) Kit provides a fast, easy and quantitative method to detect both nucleic acid and protein in the same gel. This kit uses two fluorescent dyes for detection - SYBR® Green EMSA nucleic acid gel stain for RNA or DNA detection and SYPRO® Ruby EMSA protein gel stain for protein detection. The nucleic acids and proteins are stained sequentially in the gel after electrophoresis, so there is no possibility that labeling will interfere with the protein binding being assayed. Staining only takes about 20 minutes for nucleic acid, and then about 4 hours for the subsequent protein staining.

Pierce™ Phosphoprotein Phosphate Estimation Assay Kit (Thermo Scientific™)

The Thermo Scientific Phosphoprotein Phosphate Estimation Kit enables classification and identification of proteins as phosphorylated (serine and threonine), as well as semi-quantitative assessment of the phosphorylation level.

Features of the Phosphoprotein Phosphate Estimation Kit:

Specific—measures phosphoserine (p-Ser) and phosphothreonine (p-Thr) only; does not measure phosphotyrosine (p-Tyr)
Convenient—test tube and 96-well microplate protocols included and require less than 90 minutes to perform
Semi-quantitative—calculate moles of phosphate (P) per mole of protein using the included phosvitin standard
Customizable—format is adaptable to development of specific assays for pure phosphoproteins when previously characterized standards are available

The Phosphoprotein Phosphate Estimation Assay Kit is designed to aid in characterization of the status and extent of phosphorylation of purified protein samples. The assay is based on the alkaline hydrolysis of phosphate from seryl and threonyl residues in phosphoproteins and quantification of the released phosphate with malachite green and ammonium molybdate. The assay is easily performed in 96-well microplates or test tubes and is completed in about one hour.

Applications:
• Identify proteins as containing phosphoserine or phosphothreonine phosphorylations
• Estimate the amount of pS and pT phosphorylation
• Develop specific quantitative assays for well-characterized phosphoproteins of interest

The assay can be used to identify whether a purified protein contains either phospho-serine (p-Ser) or phospho-threonine (p-Thr) as well as to estimate the level of this type of phosphorylation. For quantitation, the test protein sample is compared with specific concentrations of phosvitin, a phosphoprotein of known phosphorylation level. The alkaline hydrolysis step does not release phosphate from phospho-tyrosine (p-Tyr) residues in peptide linkage. Therefore, a negative result for an unknown purified protein preparation indicates that the protein is either (1) not a phosphoprotein or (2) is phosphorylated exclusively at tyrosine residues. In the latter case, Western blot analysis using an anti-phosphotyrosine antibody will be necessary to distinguish between these two possibilities.

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.

Related Products
Pierce™ Quantitative Peroxide Assay Kit (Aqueous)
Pierce™ Peroxide Assay Reagent A
Pierce™ Peroxide Assay Reagent B
Pierce™ Peroxide Assay Reagent C