Inconsistent Results with BCA Protein Assay Kit

June 22, 2016

University of Idaho
Biology
Research Scientist

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Ease-of-Optimization

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Company:

Thermo Scientific

Product Name:

BCA Protein Assay Kit

Catalog Number:

23225

I am currently studying the impact that BST-2 has on various enveloped viruses and one nonenveloped viruses via si RNA transfection to achieve BST-2 knockdown and TCID50 to determine viral titers. I use immunoblotting to determine whether my transfection successfully knocked down BST-2 expression. I use the Thermo Scientific BCA Protein Assay Kit to standardize protein quantities before blotting. We use this kit in particular because it works under the acidic conditions of the lysis buffer we use to extract protein.

Experimental Design and Results Summary

Application

Immunoblotting

Starting Material

Cell Lysates

Protocol Overview

Plate 10 uL of standards and samples into 96-well plate wells and apply 200 uL of working reagent per well. Mix thoroughly and incubate for at least 30 minutes. We then collect absorbency data via BMG Labtech FluoSTAR OPTIMA.

Tips

Listed 30 minute incubation is too short for results that are even close to standardized for the known concentration samples, I have a found that a 75 minute incubation yields the best results.

Results Summary

Despite our use of a recently calibrated machine and the standards included in the kit, our curve exhibited very little consistency and visible differences in absorbancy could be observed between replicates of the same standard.

Additional Notes

None

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Summary

The Good

Easy to use, accommodates very small sample sizes.

The Bad

Results between standards are visibly inconsistent, listed incubation time is too short.

The Bottom Line

The Thermo Scientific BCA Protein Assay kit is very easy to use and has a short setup time. However, inconsistent results and a longer-than-listed incubation time hurt the efficacy of this product.

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