User:Klare Lazor/Notebook/Chem-496-001/2011/10/18

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Objective

making a labeled cysteine fluorophore

Description

Use of a distant reporter group as evidence for a conformational change in a sensory receptor

  • pH increases and deprotonates
  • cysteine pka=8 our group 4 mols
  • 5mg of 5-iodoacetamido fluorescien mw 515.3g/mol
  • how much cysteine? mw= 121.16g/mol

Caculations

  • .002g * 1mol/515.3g *4mol/1mol * 121.6/1mol= 1.803 mg of cysteine / 118mg/ml= 0.015942ml of cysteine or 15.942 microliters
  • mixed in 700microliters of a pH buffer of 9.36, 5mg of 5-iodoacetamido and 0.015942 ml of cysteine
  • after 2hours for the reaction sitting, 100microliters of a sulfure containing compound was added (name : )

Reaction

  • Ph increases and deprotonates the cysteine, the deprotonated nitrogen(nucleophile) is then able to attack they carbonyl group of the 5-iodoactamido and attach.

Importance of this reaction

  • The excellent water solubility of the fluorescein 5-iodoacetamide, single isomer (M0638)

at pH 7 makes it easy to prepare green-fluorescent thiol conjugates of biomolecules. 5- Iodoacetamidofluorescein has been one of the most extensively used visible wavelength– excitable, thiol-reactive dyes for modification of proteins, nucleic acids and other biopolymers in the literature. Following conjugation to thiols, fluorescein-5- iodoacetamide can be radioiodinated. Conjugates of M0638 have a high intrinsic detectability, particularly when used with instrumentation that incorporate the 488 nm spectral line of the argon-ion laser. Furthermore, its negative charge also makes capillary electrophoretic separation of labeled adducts possible. (MGT inc. Product information sheet www.markergen.com)

  • Fluorescein (68), a polycyclic fluorophore with absorption

and fluorescence maxima in the visible region of the electromagnetic spectra (λabs 490 nm and λem 512 nm, in water), is one of the most common labels used in biological applications.

Reagents:


Image:0638.gif


Figure: 5-(Iodoacetamido)fluorescein MW= 515.3 g/mol Molecular Formula: C22H14INO6



Image:200px-Cysteine.png

Figure: Cysteine MW: 121.16g/mol

Data

  • Add data and results here...

Notes

This area is for any observations or conclusions that you would like to note.


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