Click-It Metabolic Labeling

November 30, 2015

BlueskyReddit
Colorado State University
Food Science and Human Nutrition
Graduate Student

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Quality of Results

Ease-of-Optimization

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Company:

Invitrogen

Product Name:

Click-iT® Protein Reaction Buffer Kit

Catalog Number:

C10276

The aim of this project was to detect total cellular protein synthesis in a variety of treatments using click-it chemistry. We selected this product to try as a new detection method for protein synthesis in vitro.

Experimental Design and Results Summary

Application

Gel electrophoresis, western blot, mass spectrometry

Starting Material

Total cell lysates of H4IIE rat hepatoma cells

Protocol Overview

To be able to detect newly synthesized proteins, cells were incubated with an azide moeity (does not come with kit) which couples to methionine of newly synthesized proteins. The kit comes with a series of 4 reagents that couples the protein-azide moeity with a biotin alkyne which can then be coupled to streptavidin with an attached fluorophore that can be detected via western blot.

Tips

Don't use any chemicals that may interfere with a protein concentration kit, as you need to measure protein concentration multiple times throughout the experiment.

Results Summary

The results obtained were imaged using Li-Cor Bioscience Odyssey infrared imaging system which detected the fluorophore attached to streptavidin which bound to biotin that coupled the azide-protein moeity. Results show total protein synthesis in a number of treatment groups.

Additional Notes

None

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Summary

The Good

Detects total cellular protein synthesis using relatively simple methods.

The Bad

Experiment is long with minimal breaks

The Bottom Line

Click-It metabolic labeling takes a little bit of time to optimize and experiment days are long but it's relatively cheap compared to other techniques capable of measuring protein synthesis. This kit is also very reliable. This specific experiment has been performed three times and has shown significant differences between treatment groups.

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