Global Protein Synthesis Assay Kit (FACS/Microscopy), Red Fluorescence (BN00935)

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BN00935
  • Global Protein Synthesis Assay Kit (FACS/Microscopy), Red Fluorescence (BN00935)
  • Global Protein Synthesis Assay Kit (FACS/Microscopy), Red Fluorescence (BN00935)
¥78,659.70

Description

ELISA Kit Technical ManualMSDS

Global Protein Synthesis Assay Kit (FACS/Microscopy), Red Fluorescence

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. Assay Genie's Protein Synthesis Monitoring Assay Kit 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. Our 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.

Figure: Inhibitory effect of Cycloheximide on nascent polypeptides synthesis. HeLa (10 5 cells/ ml) and Jurkat (1X10 6 cells/ml) cells respectively were pre-treated with vehicle or Cycloheximide for 30 min at 37°C. Subsequently, cells were incubated for additional 30 min with fresh aliquots of media containing either Protein Label or Protein Label and Cycloheximide. Cells were then processed and analyzed by Microscopy and FACS according to the kit protocol. (A) 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. (B) 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.

Key Information Description

Product SKU

BN00935

Size

100 assays

Kit Summary

  • Detection method- Flow Cytometry (Ex/Em 440/(540/580) nm) and Fluorescence Microscopy
  • Sample type- Adherent and suspension cells
  • Species reactivity- Mammalian
  • Applications- This assay provides a convenient and accurate procedure to measure de novo Protein synthesized in biological samples.

Detection Method

Flow Cytometry (Ex/Em (440/490)/(530/590) nm) and Fluorescence Microscopy

Species Reactivity

Eukaryotes

Applications

This assay provides a convenient and accurate procedure to measure de novo protein synthesized in biological samples.

Features and Benefits

Simple, fast, does not require lengthy incubation times

Kit Components

  • Wash Buffer (10X)
  • Fixative Solution Permeabilization Buffer (10X)
  • EZClickTM Protein Label (400X)
  • Copper Reagent (100X)
  • Fluorescent Azide (100X)
  • Reducing Agent (20X)
  • Total DNA Stain (1000X)
  • Cycloheximide (100X)

Storage Conditions

-20 °C

Shipping Conditions

Gel Pack

USAGE

For Research Use Only! Not For Use in Humans.

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