Protein Synthesis Assay Kit (Green) (ab239725)
Key features and details
- Detection method: Fluorescent
- Platform: Flow cytometer, Fluorescence microscope
- Sample type: Adherent cells, Suspension cells
Overview
-
Product name
Protein Synthesis Assay Kit (Green)
See all Protein Synthesis kits -
Detection method
Fluorescent -
Sample type
Adherent cells, Suspension cells -
Product overview
Protein Synthesis Assay Kit (ab239725) 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. -
Platform
Flow cytometer, Fluorescence microscope
Properties
-
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
-
Inhibitory effect of Cycloheximide on nascent polypeptides synthesis.
HeLa (105 cells/ ml) and Jurkat (1X106 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) Green 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 green signal is a result of Protein Label incorporation.
-
Inhibitory effect of Cycloheximide on nascent polypeptides synthesis.HeLa (105 cells/ ml) and Jurkat (1X106 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. (B) FACS analysis of negative control (white), background (Azide only, blue), positive control (Protein Label, green) and Cycloheximide-treated (pink) cell populations. Signal measured in FL-1 channel clearly shows the inhibitory effect of Cycloheximide on nascent polypeptides synthesis.
