Analysis of Small Molecular Weight Protein Samples

January 14, 2016

University of Montana
BMED
Technician

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Ease-of-Optimization

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Company:

Agilent

Product Name:

Agilent Protein 80 Kit

Catalog Number:

5067-1515

The Agilent Protein 80 kit allows for analysis of protein samples ranging is size from 5kDa-80kDa. Ten samples can be analyzed (run time) in about 45-60 minutes. Immunoprecipitated samples are denatured at 95°C for 5 minutes, allowed to cool and then loaded onto the prepared protein chip. Samples are then analyzed using the Bioanalyzer 2000. The molecular weight of our sample of interest is 18kDa and standard western blot procedure is not as sensative as we would have liked. Using this kit allowed us the sensitivity and accuarcy to interpret our results.

Experimental Design and Results Summary

Application

Protein quantification and molecular weight determination

Starting Material

Immunoprecipatated protein sample

Protocol Overview

Once the gel dye matrix, destaining and denaturing solutions are prepared the protein sample and ladder are denatured for 5 minutes and loaded onto a protein chip that has been pre loaded with the gel matrix. Total start to finish time once reagents are prepared is less than 90 minutes.

Tips

Have solutions prepared and ready to load prior to denaturing sample.

Results Summary

The results given by the Bioanalyzer 2100 software are a digital gel image similar to a coomassie stained gel, and an hlectropherogram. The hlectropherogram scan shows peaks correlating to protein bands. Quantification as well as size are listed and are +/- 10%. This is particularly useful as we often look at protein expression as a function of treatment.

Additional Notes

This analysis is independent of the actual immunoprecipatation which also must be optimized.

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Summary

The Good

Accurate size and quantification of small molecular weight proteins.

The Bad

A little time consuming in sample and reagent preparation.

The Bottom Line

If you have a protein of interest that requires quantification, the protein 80 kit provides accurate results when compared to standard western blotting.

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