Amplite® Fluorimetric Protein Quantitation Kit Orange Fluorescence

Protein quantification is an essential task in protein purification, electrophoresis, cell biology, molecular biology and other research applications. Biuret, Lowry, BCA and Bradford assays are routinely used for estimating protein concentration. However, these colorimetric assays are less sensitive, and require large sample volume to ensure accuracy. Our Amplite® Fluorimetric Protein Quantitation Kit is significantly more sensitive than existing colorimetric protein measurements, e.g., Bradford and Bicinchoninic acid (BCA) assays. Prolite™ Orange used in the kit is non-fluorescent in aqueous solution, but reacts rapidly with proteins and generates bright fluorescence. The Amplite® Fluorimetric Protein Quantitation Kit provides a simple method for quantifying protein concentration in solutions. As little as 0.1 µg/mL of BSA can be detected. The kit can be performed in a convenient 96-well or 384-well microtiter-plate format. It can be completed within 30 minutes with the fluorescence signal easily monitored. This kit has been used for (1) studying protein/protein interactions; (2) measuring column fractions after affinity chromatography; (3) estimating recovery of membrane proteins from cell extract; and (4) high-throughput screening of fusion proteins.

Example protocol

AT A GLANCE

Protocol summary

Prepare Prolite™ Orange working solution (50 µL)
Add BSA standards and/or test samples (50 µL)
Incubate at room temperature for 30 minutes
Read fluorescence intensity at Ex/Em = 485/590 nm

Important notes
Thaw all the kit components at room temperature before starting the experiment.

PREPARATION OF STOCK SOLUTION

Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.

Protein Enhancer stock solution (500X):
Add 100 µL of Protein Enhancer Buffer (Component E) into the vial of Protein Enhancer (Component C). Note: 20 µL of Protein Enhancer stock solution (500X) is enough for make 10 mL working solution. Unused Protein Enhancer stock solution (500X) should be stored in single use aliquots at -20 ^oC.

PREPARATION OF STANDARD SOLUTION

BSA standard

For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11105

Note: Standards and test samples should be prepared in Protein Enhancer working solution.

PREPARATION OF WORKING SOLUTION

1. Protein Enhancer working solution:
Add 20 µL of Protein Enhancer stock solution (500X) into 10 mL of Assay Buffer (Component D) and mix them well.

2. Prolite™ Orange Working Solution:
Add 10 µL of Prolite™ Orange (Component A) into 5 mL of Assay Buffer (Component D) and mix them well. Note: 5 mL of Prolite™ Orange working solution is enough for 1 plate. Prepare enough Prolite™ Orange working solution fresh for each experiment.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of BSA standards and test samples in a solid black 96-well microplate. BS = BSA standard (BS1-BS7); BL = blank control; TS = test sample.

BL	BL	TS	TS
BS1	BS1	...	...
BS2	BS2	...	...
BS3	BS3
BS4	BS4
BS5	BS5
BS6	BS6
BS7	BS7

Table 2. Reagent composition for each well

BSA Standard	Blank Control	Test Sample
Serial Dilutions: 50 µL	Protein Enhancer working solution: 50 µL	50 µL

Protein assay

Add 50 µL of BSA standard, blank control, and test samples (See table 1 and 2) into a solid black 96-well plate. Note: For a 384-well plate, add 20 µL of samples.
Add 50 µL/well of Prolite™ Orange working solution into BSA standard, blank control, and test samples to make the total assay volume of 100 µL/well. Note: For a 384-well plate, add 20 µL of Prolite™ Orange working solution into each well.
Incubate the reaction for 10 to 30 minutes at 37 ^oC, protected from light.
Monitor the the fluorescence increase with a fluorescence plate reader at Ex/Em = 485/590 nm.

References

View all 27 references: Citation Explorer

Use of anchor protein modules in fluorescence polarisation aptamer assay for ochratoxin A determination
Authors: Samokhvalov, A. V.; Safenkova, I. V.; Eremin, S. A.; Zherdev, A. V.; Dzantiev, B. B.
Journal: Anal Chim Acta (2017): 80-87

Quantification of Membrane Protein Self-Association with a High-Throughput Compatible Fluorescence Assay
Authors: Li, J.; Qiu, X. J.
Journal: Biochemistry (2017): 1951-1954

Dual Amplification Fluorescence Assay for Alpha Fetal Protein Utilizing Immunohybridization Chain Reaction and Metal-Enhanced Fluorescence of Carbon Nanodots
Authors: Xu, D. D.; Liu, C.; Li, C. Y.; Song, C. Y.; Kang, Y. F.; Qi, C. B.; Lin, Y.; Pang, D. W.; Tang, H. W.
Journal: ACS Appl Mater Interfaces (2017): 37606-37614

Tryptophan fluorescence quenching as a binding assay to monitor protein conformation changes in the membrane of intact mitochondria
Authors: Akbar, S. M.; Sreeramulu, K.; Sharma, H. C.
Journal: J Bioenerg Biomembr (2016): 241-7

Label-free fluorescence assay for protein kinase based on peptide biomineralized gold nanoclusters as signal sensing probe
Authors: Song, W.; Wang, Y.; Liang, R. P.; Zhang, L.; Qiu, J. D.
Journal: Biosens Bioelectron (2015): 234-40

Page updated on December 17, 2024

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