Protein Synthesis Assay Kit (Red) (ab235634)
Key features and details
- Detection method: Fluorescent
- Platform: Flow cytometer, Fluorescence microscope
- Sample type: Adherent cells, Suspension cells
Overview
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Product name
Protein Synthesis Assay Kit (Red)
See all Protein Synthesis kits -
Detection method
Fluorescent -
Sample type
Adherent cells, Suspension cells -
Product overview
Global Protein Synthesis Assay Kit ab235634 utilizes a novel and robust chemical method based on an alkyne containing and cell-permeable analog of puromycin, O-Propargyl-puromycin (OP-puro). Once inside the cell, OP-puro stops translation by forming covalent conjugates with 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. Unlike methionine analogs, OP-puro does not require methionine-free conditions and can be used to label nascent proteins directly in the cell culture. The kit provides sufficient materials for 100 assays to detect nascent proteins synthesized under various physiological conditions, and Cycloheximide, an inhibitor of protein synthesis that serves as an experimental control.
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Notes
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 chock, 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 treatment or environmental changes are important tools in assesment of cytotoxicity.
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Platform
Flow cytometer, Fluorescence microscope
Properties
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Storage instructions
Store at -20°C. Please refer to protocols. -
Components 100 tests Wash Buffer (10X) 1 x 25ml Fixative Solution 1 x 10ml Permeabilization Buffer (10X) 1 x 25ml Protein Label (400X) 1 x 25µl Copper Reagent (100X) 1 x 100µl Fluorescent Azide (100X) 1 x 100µl Reducing Agent (20X) 1 x 500µl Total DNA Stain (1000X) 1 x 10µl Cycloheximide (100X) 1 x 10µl -
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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Analysis of protein biosynthesis in presence of Cycloheximide.
Jurkat cells (1X106 cells/mL) were pre-incubated either with vehicle or 1X Cycloheximide for 30 minutes at 37°C incubator to suppress protein synthesis followed media exchange. Cells were then incubated either with culture medium alone (white) or 1X Protein Label (red) or 1X Cycloheximide (green) for 30 minutes in the cell culture incubator. The cells were processed for protein synthesis detection by FACS according to the included protocol. Fluorescence measured in FL-2 channel clearly shows the inhibitory effect of Cycloheximide on nascent poppypeptides synthesis.
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Inhibitory effect of Cycloheximide on nascent polypeptides synthesis.Red fluorescence (upper panel) corresponds to de novo synthesized polypeptides whereas bottom panel shows the inhibitory effect of Cycloheximide on protein biosynthesis. Nuclear staining in both panels confirms that red signal is a result of Protein Label incorporation.
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FACS analysis of the inhibitory effect of Cycloheximide.FACS analysis of negative control (white), positive control (Protein Label, red) and Cycloheximide-treated (green) cell populations. Signal measured in FL-2 channel clearly shows the inhibitory effect of Cycloheximide on nascent polypeptides synthesis.
