Mar 10, 2023
Protein Concentration Determination using Qubit 4 Fluorometer
This protocol is a draft, published without a DOI.
- 1University of Illinois at Urbana-Champaign
Protocol Citation: Steven J Burgess 2023. Protein Concentration Determination using Qubit 4 Fluorometer. protocols.io https://protocols.io/view/protein-concentration-determination-using-qubit-4-cqw2vxge
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 10, 2023
Last Modified: August 14, 2023
Protocol Integer ID: 78522
Keywords: protein quantification, qubit, soybean, leaf tissue
Abstract
Procedure for quantification of protein concentration using a Qubit 4 Fluorometer. The procedure follows the manufacturer's instructions, this version is adapted for use with samples that have been extracted in protein extraction buffer as part of processing soybean leaf tissue.
Consult the manual for further details:
Image Attribution
Image reproduced from ThermoFisher Scientific website https://www.thermofisher.com/us/en/home/industrial/spectroscopy-elemental-isotope-analysis/molecular-spectroscopy/fluorometers/qubit/qubit-fluorometer.html
Materials
Before start
This protocol assumes protein samples have been resuspended in 1x protein extraction buffer (62.5 mM Tris-HCl (pH 6.8), 2 % SDS (w/v); 10 % glycerol (v/v); 2.5% 2-mercaptoethanol (v/v) ) and the dilution factors were determined in relation to processing of 3 size 7 Humboldt leaf disks resuspended in 450 µL of buffer. Preliminary tests should be made when working with different samples or volumes
Create Working Solution for Analysis
Create Working Solution for Analysis
Create a working solution of Qubit assay buffer by diluting the reagent 1:200 in the provided buffer.
Note
The final volume in each tube must be 200 µL. Each standard tube requires 190 µL of Qubit working solution, and each sample tube requires anywhere from 180–199 µL. Therefore prepare a sufficient Qubit working solution to accommodate all standards and samples.
Create Sample Dilution for Analysis
Create Sample Dilution for Analysis
15m 6s
15m 6s
Dilute sample 1:50 (196 µL dH2O + 4 µL sample)
Note
After taking into consideration the Qubit dilution factor (1:20; 10 μL sample + 190 μL qubit assay buffer) the sample being analyzed has been diluted 1:1000. This should give a value in the linear range for qubit (1.25-25 μg/mL).
The dilution is also important to reduce the impact of SDS and 2-mercaptoethanol on quantification. The concentration of SDS must be >0.2 %, and 2-mercaptoethanol > 20 mM in the 10 μL sample added to the assay. The concentration of SDS in PEB is 2 %, and 2-mercaptoethanol 335 mM, so diluted 1:50 yields [SDS] 0.04 % and [2-mercaptoethanol] 6.7 mM, which is in the acceptable range.
Add 190 µL of Qubit working solution to a fresh Qubit assay tube, one for each sample to be analyzed (including the three protein standards)
Add 10 µL of protein standard to the appropriate tube and mix by vortexing 00:00:03 s.
3s
Add 10 µL of diluted sample to the appropriate tube and mix by vortexing 00:00:03 s.
3s
Allow samples to incubate at room temperature for 00:15:00
15m
Measure protein sample concentration using the Qubit, following the instructions on the machine (i.e. start by measuring the standard curve).
Note
Remember to adjust measured values according to the dilution factor applied. In this example, after taking into consideration the Qubit dilution factor (1:20; 10 µL sample + 190 µL Qubit assay buffer) the sample has been diluted 1:1000
Equipment
Qubit™ 4 Fluorometer, with WiFi
NAME
Fluorometer
TYPE
Invitrogen
BRAND
Q33238
SKU
LINK