Amplite® Fluorimetric Fluorescamine Protein Quantitation Kit *Blue Fluorescence*
Fluorescamine is intrinsically non-fluorescent but reacts rapidly with primary aliphatic amines, including those in peptides and proteins, to yield a blue-green-fluorescent derivative. The Amplite® fluorescamine protein assay kit provides a simple method for quantifying protein concentration in solutions. This Amplite® fluorescamine protein assay kit can be performed in a convenient 96-well or 384-well microtiter-plate format and easily adapted to automation with no separation steps required. The assay can be completed within 30 minutes. With the Amplite® fluorescamine protein assay kit, as little as 3 ug/mL of BSA can be detected.
Example protocol
AT A GLANCE
Protocol summary
- Prepare fluorescamine working solution (25 µL)
- Add BSA standards or test samples (75 µL)
- Incubate at room temperature for 5 - 30 minutes
- Read fluorescence intensity at Ex/Em = 380/470 nm
Important notes
Thaw all the kit components at room temperature before starting the experiment.
PREPARATION OF STANDARD SOLUTION
BSA standard
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11100
For convenience, use the Serial Dilution Planner: https://www.aatbio.com/tools/serial-dilution/11100
Dilute the appropriate amount of BSA Standard 1 mg/mL (Component C) into PBS by performing 1:2 serial dilutions to get serial dilutions of BSA standard (BS7 - BS1).
PREPARATION OF WORKING SOLUTION
Add the whole content of DMSO (Component B) into the bottle of Fluorescamine (Component A), and mix well. Note: 2.5 mL of fluorescamine working solution is enough for 1 plate.
SAMPLE EXPERIMENTAL PROTOCOL
Table 1. Layout of BSA standards and test samples in a solid black 96-well microplate. BS= BSA Standards (BS1 - BS7, 1.563 to 100 µg/mL), BL=Blank Control, TS=Test Samples.
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.
Well | Volume | Reagent |
BS1 - BS7 | 75 uL | Serial Dilution (1.563 to 100 ug/mL) |
BL | 75 uL | PBS |
TS | 75 uL | Test Sample |
- Prepare BSA standards (BS), blank controls (BL), and test samples (TS) according to the layout provided in Tables 1 and 2. For a 384-well plate, use 30 µL of reagent per well instead of 75 µL.
- Add 25 µL of fluorescamine working solution to each well of BSA standard, blank control, and test samples to make the total assay volume of 100 µL/well. For a 384-well plate, add 10 µL of fluorescamine working solution into each well instead, for a total volume of 40 µL/well.
- Incubate the reaction at room temperature for 5 to 30 minutes, protected from light.
- Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 380/470 nm.
Citations
View all 1 citations: Citation Explorer
Covalent vaccination with Trypanosoma cruzi Tc24 induces catalytic antibody production
Authors: Gunter, Sarah M and Versteeg, Leroy and Jones, Kathryn M and Brian, Keegan P and Strych, Ulrich and Bottazzi, Maria Elena and Hotez, Peter J and Brown, Eric L
Journal: Parasite immunology (2018): e12585
Authors: Gunter, Sarah M and Versteeg, Leroy and Jones, Kathryn M and Brian, Keegan P and Strych, Ulrich and Bottazzi, Maria Elena and Hotez, Peter J and Brown, Eric L
Journal: Parasite immunology (2018): e12585
References
View all 41 references: Citation Explorer
Fluorescence of aromatic amines and their fluorescamine derivatives for detection of explosive vapors
Authors: Eastwood D, Fern and ez C, Yoon BY, Sheaff CN, Wai CM.
Journal: Appl Spectrosc (2006): 958
Authors: Eastwood D, Fern and ez C, Yoon BY, Sheaff CN, Wai CM.
Journal: Appl Spectrosc (2006): 958
Fluorimetric determination of histamine in fish using micellar media and fluorescamine as labelling reagent
Authors: Adamou R, Coly A, Douabale SE, Saleck ML, Gaye-Seye MD, Tine A.
Journal: J Fluoresc (2005): 679
Authors: Adamou R, Coly A, Douabale SE, Saleck ML, Gaye-Seye MD, Tine A.
Journal: J Fluoresc (2005): 679
Determination of 4-amino-m-cresol and 5-amino-o-cresol by high performance liquid chromatography and fluorescence derivatization using fluorescamine
Authors: Eggenreich K, Zach E, Beck H, Wintersteiger R.
Journal: J Biochem Biophys Methods (2004): 35
Authors: Eggenreich K, Zach E, Beck H, Wintersteiger R.
Journal: J Biochem Biophys Methods (2004): 35
Chiral separation of fluorescamine-labeled amino acids using microfabricated capillary electrophoresis devices for extraterrestrial exploration
Authors: Skelley AM, Mathies RA.
Journal: J Chromatogr A (2003): 191
Authors: Skelley AM, Mathies RA.
Journal: J Chromatogr A (2003): 191
LC determination of aminoglutethimide enantiomers as dansyl and fluorescamine derivatives in tablet formulations
Authors: Cesur N, Apak TI, Aboul-Enein HY, Ozkirimli S.
Journal: J Pharm Biomed Anal (2002): 487
Authors: Cesur N, Apak TI, Aboul-Enein HY, Ozkirimli S.
Journal: J Pharm Biomed Anal (2002): 487
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