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Amplite® Fluorimetric Fluorescamine Protein Quantitation Kit *Blue Fluorescence*

BSA dose response was measured on a solid black 96-well plate with Amplite® Fluoremetric Fluorescamine Protein Quantitation Assay Kit.
BSA dose response was measured on a solid black 96-well plate with Amplite® Fluoremetric Fluorescamine Protein Quantitation Assay Kit.
BSA dose response was measured on a solid black 96-well plate with Amplite® Fluoremetric Fluorescamine Protein Quantitation Assay Kit.
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Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

OverviewpdfSDSpdfProtocol


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.

Platform


Fluorescence microplate reader

Excitation380
Emission470
Cutoff420
Recommended plateSolid black

Components


Example protocol


AT A GLANCE

Protocol summary

  1. Prepare  fluorescamine working solution (25 µL)
  2. Add BSA standards or test samples (75 µL)
  3. Incubate at room temperature for 5 - 30 minutes
  4. 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

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.

BLBLTSTS
BS1BS1......
BS2BS2......
BS3BS3  
BS4BS4  
BS5BS5  
BS6BS6  
BS7BS7  

Table 2. Reagent composition for each well.

WellVolumeReagent
BS1 - BS775 uLSerial Dilution (1.563 to 100 ug/mL)
BL75 uLPBS
TS75 uLTest Sample
  1. 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.

  2. 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.

  3. Incubate the reaction at room temperature for 5 to 30 minutes, protected from light.

  4. Monitor the fluorescence increase with a fluorescence plate reader at Ex/Em = 380/470 nm.

Images


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

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
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
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
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
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
Spectrofluorimetric determination of vigabatrin and gabapentin in urine and dosage forms through derivatization with fluorescamine
Authors: Belal F, Abdine H, Al-Majed A, Khalil NY.
Journal: J Pharm Biomed Anal (2002): 253
A comparison of fluorescamine and naphthalene-2,3-dicarboxaldehyde fluorogenic reagents for microplate-based detection of amino acids
Authors: Bantan-Polak T, Kassai M, Grant KB.
Journal: Anal Biochem (2001): 128
Detection, quantitation, and identification of residual aminopenicillins by high-performance liquid chromatography after fluorescamine derivation
Authors: Hong C, Kondo F.
Journal: J Food Prot (2000): 1421
Determination of taurine in plasma by capillary zone electrophoresis following derivatisation with fluorescamine
Authors: Kelly MT, Fabre H, Perrett D.
Journal: Electrophoresis (2000): 699
Characterisation of the binding interaction between poly(L-lysine) and DNA using the fluorescamine assay in the preparation of non-viral gene delivery vectors
Authors: Read ML, Etrych T, Ulbrich K, Seymour LW.
Journal: FEBS Lett (1999): 96