Global Protein Synthesis Assay Kit (ab273286)
Key features and details
- Assay type: Quantitative
- Detection method: Fluorescent
- Platform: Microplate
- Sample type: Adherent cells, Suspension cells
Overview
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Product name
Global Protein Synthesis Assay Kit
See all Protein Synthesis kits -
Detection method
Fluorescent -
Sample type
Adherent cells, Suspension cells -
Assay type
Quantitative -
Assay duration
Multiple steps standard assay -
Product overview
Global Protein Synthesis Assay Kit (ab273286) utilizes a novel chemical method based on alkyne analog of puromycin, O-Propargyl-puromycin (OP-puro). OP-puro stops translation by forming covalent conjugates with the nascent polypeptide chains. Truncated polypeptides are rapidly turned over by the proteasome and can be detected based on a click reaction with the fluorescent azide (Ex/Em = 494/521 nm). OP-puro does not require methionine-free conditions and can be used to label nascent proteins directly in the cell culture medium.
This kit provides sufficient materials for 100 simple and specific assays to detect nascent proteins synthesized under various physiological conditions in real-time, and in the presence of Cycloheximide, an inhibitor of protein synthesis that serves as a control.
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Platform
Microplate
Properties
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Storage instructions
Store at -20°C. Please refer to protocols. -
Components 100 tests Copper Reagent (100X) 1 x 100µl Cycloheximide (100X) 1 x 10µl Fixative Solution 1 x 10ml Fluorescent Azide (100X) 1 x 100µl Permeabilization Buffer (10X) 1 x 25ml Protein Label (400X) 1 x 25µl Reducing Agent (20X) 1 x 500µl Total DNA Stain (1000X) 1 x 20µl Wash Buffer (10X) 1 x 25ml -
Relevance
Cells generate a complete set of proteins during division. Protein synthesis is a tightly regulated process and many critical controls in gene expression occur at the level of translation to ensure that production of specific cellular proteins is quickly turned on/off under specific conditions (heat shock, starvation, etc.). Protein synthesis is essential in cell growth, proliferation, signaling, differentiation or death; therefore, the identity and amount of the synthesized proteins are critical parameters in determining the physiological state of the cell. Methods enabling detection and characterization of nascent proteins, or changes in spatial and temporal protein expression/degradation patterns during disease, drug treatments or environmental changes are important tools in assessment of cytotoxicity.
Images
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Metabolic labeling of protein on proliferating HeLa cells
Metabolic labeling of protein on proliferating HeLa cells. 1 x 105 cells incubated overnight with fresh aliquots of media containing Protein Label: Upper panel corresponds green fluorescence of de novo synthesized peptides. The lower panel shows panel cells treated with 1X Cycloheximide. Nuclear staining in both panels confirms that green signal is a result of Protein Label incorporation.
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Plate reader analyses on Jurkat cellsJurkat cells: plate reader analyses of controls and Cycloheximide treatment; Avg fluorescence +/- standard deviation plotted for 3 replicates per condition.
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Azide Fluorescence Curve in 0-0.1 X rangeTypical standard curve – data provided for demonstration purposes only. A new standard curve must be generated for each assay performed.
This is reference data and it should not be used to interpret actual results. Your data will depend on the cell type and tested compound.
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Fluorescence Azide Curve of Jurkat cellsTypical standard curve – data provided for demonstration purposes only. A new standard curve must be generated for each assay performed.
Fluorescence azide curve of Jurkat cells prepared for this assay. Detection limit corresponds to about 31,250 of Jurkat cells per well.
