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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.
Ordering information
Price ()
Catalog Number11100
Unit Size
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Additional ordering information
Telephone1-408-733-1055
Fax1-408-733-1304
Emailsales@aatbio.com
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Molecular weight278.26
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
UNSPSC12171501

OverviewpdfSDSpdfProtocol


Molecular weight
278.26
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


Component A: Fluorescamine1 bottle
Component B: DMSO1 bottle (5 mL)
Component C: BSA Standard 1 vial (0.5 mL)

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.

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
  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.

Calculators


Common stock solution preparation

Table 1. Volume of appropriate solvent needed to reconstitute specific mass of Amplite® Fluorimetric Fluorescamine Protein Quantitation Kit *Blue Fluorescence* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.

0.1 mg0.5 mg1 mg5 mg10 mg
1 mM359.376 µL1.797 mL3.594 mL17.969 mL35.938 mL
5 mM71.875 µL359.376 µL718.752 µL3.594 mL7.188 mL
10 mM35.938 µL179.688 µL359.376 µL1.797 mL3.594 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
/=x=

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