Amplite™ Fluorimetric Protein Quantitation Kit *Orange Fluorescence*

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<p>BSA, chicken-egg ovalbumin, porcine thyroglobulin dose response was measured at Ex/Em 485/590 in a 96-well black plate with the Amplite™ Fluoremetric Protein Quantitation Kit. As low as 0.1 ug/mL of BSA can be detected.</p>
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500 tests 11105 $195


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Additional Ordering Information
Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
International: See distributors





Overview

PlatformsFluorescence microplate reader
Storage Freeze (<-15 °C)
Minimize light exposure
Category Protein Biochemistry
General proteins
Related
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 at Ex/Em = 485/590 nm. 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.




Protocol


Quick Preview

This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

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

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

Key parameters
Instrument:Fluorescence microplate reader
Excitation:485 nm
Emission:590 nm
Cutoff:570 nm
Recommended plate:Solid black
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

  1. 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.

  2. 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.

  3. Incubate the reaction for 10 to 30 minutes at 37 oC, protected from light.

  4. Monitor the the fluorescence increase with a fluorescence plate reader at Ex/Em = 485/590 nm.
Example data analysis and figures

Figure 1.

BSA, chicken-egg ovalbumin, porcine thyroglobulin dose response was measured at Ex/Em 485/590 in a 96-well black plate with the Amplite™ Fluoremetric Protein Quantitation Kit. As low as 0.1 ug/mL of BSA can be detected.

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.





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References

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

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

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

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

Ag@SiO2-entrapped hydrogel microarray: a new platform for a metal-enhanced fluorescence-based protein assay
Authors: Jang, E.; Kim, M.; Koh, W. G.
Journal: Analyst (2015): 3375-83

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

Budded baculoviruses as a tool for a homogeneous fluorescence anisotropy-based assay of ligand binding to G protein-coupled receptors: the case of melanocortin 4 receptors
Authors: Veiksina, S.; Kopanchuk, S.; Rinken, A.
Journal: Biochim Biophys Acta (2014): 372-81

Characterization of G protein-coupled receptors by a fluorescence-based calcium mobilization assay
Authors: Caers, J.; Peymen, K.; Suetens, N.; Temmerman, L.; Janssen, T.; Schoofs, L.; Beets, I.
Journal: J Vis Exp (2014): e51516

Cleavage of pro-tumor necrosis factor alpha by ADAM metallopeptidase domain 17: a fluorescence-based protease assay cleaves its natural protein substrate
Authors: Zhang, C.; Zheng, L.; Nurnberg, J.; Vacari, B. M.; Zhou, J.; Wang, Y.
Journal: Anal Biochem (2014): 14-9

A fluorescence-based thermal shift assay identifies inhibitors of mitogen activated protein kinase kinase 4
Authors: Krishna, S. N.; Luan, C. H.; Mishra, R. K.; Xu, L.; Scheidt, K. A.; Anderson, W. F.; Bergan, R. C.
Journal: PLoS One (2013): e81504


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Safety Data Sheet (SDS)


Certificate of Analysis