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Portelite™ Fluorimetric Protein Quantitation Kit *Optimized for CytoCite™ and Qubit™ Fluorometers*

Serial dilutions of BSA, chicken-egg ovalbumin, porcine thyroglobulin were measured at Ex/Em 485/590 nm using Portelite™ Fluorimetric Protein Quantitation Kit *Optimized for CytoCite™ and Qubit™ Fluorometers* with Qubit® Fluorometer. As low as 50 ng/mL of protein can be detected.
Serial dilutions of BSA, chicken-egg ovalbumin, porcine thyroglobulin were measured at Ex/Em 485/590 nm using Portelite™ Fluorimetric Protein Quantitation Kit *Optimized for CytoCite™ and Qubit™ Fluorometers* with Qubit® Fluorometer. As low as 50 ng/mL of protein can be detected.
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Catalog Number11109
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OverviewpdfSDSpdfProtocol


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 Portelite™ 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 Portelite™ Fluorimetric Protein Quantitation Kit provides a simple method for quantifying protein concentration in solutions. The assay has dynamic range from 12.5 ug/mL to 5 mg/mL of BSA. The kit is optimized for Cytocite™ and Qubit™ fluorometers. It can be 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.

Platform


Qubit Fluorometer

Excitation480 nm
Emission510-580 nm
Instrument specification(s)0.2 mL, thin-wall PCR tube

CytoCite Fluorometer

Excitation480 nm
Emission510-580 nm
Instrument specification(s)0.2 mL, thin-wall PCR tube

Components


Component A: Prolite™ Orange 1 vial (300 µL)
Component B: BSA Standard 1 1 vial (1 mL)
Component C: BSA Standard 2 1 vial (1 mL)
Component D: BSA Standard 3 1 vial (1 mL)
Component E: Sample Dilution Buffer1 bottle (60 mL)

Example protocol


AT A GLANCE

Protocol summary

  1. Prepare and add BSA standards or test samples (10 µL)
  2. Prepare and add Prolite™ Orange working solution (190 µL) in a 0.2 mL PCR tube (Cat# CCT100)
  3. Incubate at room temperature for 15 minutes
  4. Monitor fluoroscence with CytoCite™ or Qubit fluorometer

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

PREPARATION OF WORKING SOLUTION

Prolite™ Orange working solution:
Add 5 µL of Prolite™ Orange (200X) (Component A) to 995 µL of Sample Dilution Buffer (Component E) and mix them well. Note: Do not mix the working solution in a glass container. Note: Prepare the amount as needed.

SAMPLE EXPERIMENTAL PROTOCOL

This protocol is generated based upon Qubit® Fluorometer.

Run protein assay

  1. Add 190 µL/well of Prolite™ Orange working solution into each tube.

  2. Add 10 µL BSA standards (Component B, C, D) or 10 µL samples into the 190 µL Prolite™ Orange working solution tube to make the final assay volume 200 µL/tube.

  3. Incubate the reaction at room temperature for 15 minutes. Note: Protect the samples from light and avoid holding the samples in hands.

  4. Insert the samples into CytoCite™ and monitor the fluorescence with Green fluorescent channel. Follow the procedure appropriate for CytoCite™ Fluorometer. See the link below for detailed instructions:
    https://devices.aatbio.com/documentation/user-manual-for-cytocite-fluorometer

Brief protocol for Qubit® fluorometer

  1. Press Protein on the Home screen of the Qubit® Home screen and proceed to press Read standards.

  2. Insert each of the 3 tubes contains standards into the sample chamber.

  3. Close the lid and press Read standards.

  4. The instrument displays the results and generates calibration curve.

  5. Press Run samples and select sample volume to 10 µL.

  6. Insert the sample tube into the sample chamber.

  7. Close the lid and press Read tube.

  8. The instrument displays the results on the assay screen. The top value is the original sample concentration and bottom value is the diluted concentration.

PREPARATION OF STANDARD SOLUTION

For CytoCite™ Fluorometer assays, you have the choice to run a calibration with your own protein standards. Here is a brief protocol to generate a customized protein standard curve.

  1. Prepare a protein solution of 400µg/ml (400 ng/µL) in PBS buffer.

  2. Perform 1:2 serial dilution with PBS buffer to get 200, 100, 50, 25, 12.5 ng/µl serial standard dilutions.

  3. Add 190 µL of Prolite™ Orange working solution into a 0.2 mL PCR tube.

  4. Add 10 µL standards or 10 µL samples into each tube.

  5. Incubate the reaction at room temperature for 15 minutes.

  6. Insert the samples into CytoCite™ and monitor the fluorescence with Green fluorescent channel.

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
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
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
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
Development of a fluorescence intensity assay for the mitotic serine/threonine protein kinase Aurora-A
Authors: Slatter, A. F.; Campbell, S.; Angell, R. M.
Journal: J Biomol Screen (2013): 219-25
Direct comparison of the histidine-rich protein-2 enzyme-linked immunosorbent assay (HRP-2 ELISA) and malaria SYBR green I fluorescence (MSF) drug sensitivity tests in Plasmodium falciparum reference clones and fresh ex vivo field isolates from Cambodia
Authors: Chaorattanakawee, S.; Tyner, S. D.; Lon, C.; Yingyuen, K.; Ruttvisutinunt, W.; Sundrakes, S.; Sai-gnam, P.; Johnson, J. D.; Walsh, D. S.; Saunders, D. L.; Lanteri, C. A.
Journal: Malar J (2013): 239