Amplite® Fluorimetric Protein Quantitation Kit *Orange Fluorescence*
Overview | ![]() ![]() |
Platform
Fluorescence microplate reader
Excitation | 485 nm |
Emission | 590 nm |
Cutoff | 570 nm |
Recommended plate | Solid black |
Components
Component A: 500X Prolite™ Orange | 1 vial (50 µL) |
Component B: BSA Standard (1 mg/mL) | 1 vial (0.5 mL) |
Component C: Protein Enhancer | 1 vial |
Component D: Assay Buffer | 1 bottle (50 mL) |
Component E: Protein Enhancer Buffer | 1 vial (100 µL) |
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
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
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.
Product family
References
Authors: Samokhvalov, A. V.; Safenkova, I. V.; Eremin, S. A.; Zherdev, A. V.; Dzantiev, B. B.
Journal: Anal Chim Acta (2017): 80-87
Authors: Li, J.; Qiu, X. J.
Journal: Biochemistry (2017): 1951-1954
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
Authors: Akbar, S. M.; Sreeramulu, K.; Sharma, H. C.
Journal: J Bioenerg Biomembr (2016): 241-7
Authors: Song, W.; Wang, Y.; Liang, R. P.; Zhang, L.; Qiu, J. D.
Journal: Biosens Bioelectron (2015): 234-40
Authors: Caers, J.; Peymen, K.; Suetens, N.; Temmerman, L.; Janssen, T.; Schoofs, L.; Beets, I.
Journal: J Vis Exp (2014): e51516
Authors: Veiksina, S.; Kopanchuk, S.; Rinken, A.
Journal: Biochim Biophys Acta (2014): 372-81
Authors: Zhang, C.; Zheng, L.; Nurnberg, J.; Vacari, B. M.; Zhou, J.; Wang, Y.
Journal: Anal Biochem (2014): 14-9
Authors: Slatter, A. F.; Campbell, S.; Angell, R. M.
Journal: J Biomol Screen (2013): 219-25
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
Application notes
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