TN 236 Thermo Fisher Qubit™ Protein BR Assay Performance Data

Introduction

DeNovix DS-11 Series and QFX Fluorometers enable the accurate and specific quantification of nucleic acids, proteins and other biomolecules through fluorescence measurements. This technical note presents typical performance data obtained on the DeNovix QFX using the Thermo Fisher Qubit™ Protein Broad Range (BR) Assay. 

DeNovix fluorometers utilize a proprietary optical core and a versatile set of four fluorescence channels for the excitation and emission detection of a wide range of fluorophores. These channels enable fluorescent quantification of protein as well as dsDNA, RNA, and ssDNA using a range of different assay kits from a variety of manufacturers.

Protein Quantification Using the Qubit™ Protein BR Assay

The Thermo Fisher Qubit™ Protein BR Assay is designed for measurement ranges between 1 mg and 200 mg total mass, which translates to 0.1 mg/mL and 20 mg/mL of sample. The assay is designed to work with sample volumes of 10 µL or 20 µL combined with other kit components, totaling to 200 µL in the measurement tube.

According to manufacturer instructions, for best performance, adding the larger volume of sample is preferred, but this will depend upon how much total sample is available for measurement. 10 µL samples will still work, but more reliable performance is achieved when using 20 µL samples where appropriate.

Materials and Methods

The Thermo Fisher Qubit™ Protein BR assay kit (Thermo Fisher Scientific cat #A50668) was used to perform the assay. Samples were measured in thin-walled, clear UV-transparent 0.5 mL PCR tubes (DeNovix cat #TUBE-PCR-0.5-500).

Performance data for the Thermo Fisher Qubit™ Protein BR quantification assay was obtained on a Thermo Fisher Qubit™ 4.0 fluorometer and a DeNovix QFX Fluorometer. Each assay was prepared as described in the manufacturer’s protocol. Samples were mixed and incubated at room temperature for 10 minutes. Three replicate measurements were taken for each sample.

A series of dilutions was gravimetrically prepared in HPLC grade water from the Thermo Fisher Qubit™ Protein BR assay standards. Each sample was prepared to fall within the total mass limits of the assay. For each assay, 10 or 20 μL of each prepared sample was added to 150 µL or 160 μL assay buffer and combined with 30 µL of the Protein BR reagent according to the instructions. Initial sample concentrations were between 0.1 mg/mL and 20 ng/μL based on the limits of the assay.

Summary

The data presented in this technical document shows that the DeNovix QFX Fluorometer enables sensitive and accurate quantitation of proteins across the full published range of the Thermo Fisher Qubit™ Protein BR assay. The DeNovix QFX protein quantitation performance is equivalent to Thermo Fisher Qubit™ performance across the full dynamic range and offers greater flexibility in the choice of assays and fluorophores.

6-JAN-2025