User:Klare Lazor/Notebook/Chem-496-001/2012/02/08

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Objective

tagging bsa continued

  • mixing BSA with dye in buffer solution prepared yesterday to prepare a control
  • Next tuesday fluorescene will be tested, if successful attachment of dye to nano particle will be completed tuesday and then compared with the control on wednesday

Description

  • Use of a distant reporter group as evidence for a conformational change in a sensory receptor
  • pH increases and deprotonates
  • cysteine pka=8
  • 3mg of 5-iodoacetamido fluorescien mw 515.3g/mol (4 Fold Excess)
  • how much cysteine? mw= 121.16g/mol
    • 35 cysteine in BSA

Caculations

  • 35(121.16g/mol)= 4240.6 g/mol of cysteine
  • (4240.6 g/mol of cysteine)/(67,000g/mol of BSA)= 0.06329 (mass ratio)
  • (0.06329)*(0.00495 grams amount of BSA in stock)= 0.000313g/50mL of solution
  • (0.000313g/50mL)/(121.16 g/mol)= 2.5E-6mol/mL
  • (2.5E-6 mol/mL)/ (50mL)= 5.172E-7 mols of cysteine per mL solution
    • Whatever mLs of BSA we use in sample(2.767mL) X 5.172E-7mols of cysteine per mL solution=0.000001 mols of cysteine
    • To determine amount of 5-iodoacetamido fluorecien
      • .000001 mols of cytiene x (4mol of dye/1 mol cysteine) x 515.3g/mol= 0.0029 grams of 5-iodoacetamido fluoreciene


Calculations for buffer Solution

  • pka= 7.21 for phosphate
  • ph=pka+log[salt]/[acid]
  • 7.21=7.21 +log[salt]/[acid]
  • [salt]/[acid]= 1
  • so any concentration as long as they have a ratio of 1, we use 0.05mols/Liter
  • may have to increase the amount of Na2HPO4 because it is hydrated
    • 141.98g/mol Na2HPO4- * 0.050 mol/L = 7.099 g/L x .25 L =1.7747g ACTUALLY ADDED ADDITIONAL 2 GRAMS
    • 137.99g/mol x 0.05 mol/L = 6.89 x .25 L = 1.725 g


Procedure

  • mixed in 406 microliters of a pH buffer of 7, 2.9 mg of 5-iodoacetamido and 2.776 mL BSA from Stock
    • Ideal ph is between 7 and 8
  • after 2hours let reaction sit
  • place in dialysis bag in beaker with water with stir bar
    • replace water twice tom, and once daily till next tuesday
  • New concentration of BSA 13.6┬ÁM

Whats going on

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

Data

  • .0026grams of dye actually used
  • wrap in tin foil so light does not kill the fluorescence

Image:Photoooo.JPG

  • it was noted that dye in water fluoresced less than in buffer due to the distilled water was probably not ph 7
  • The reason why we tagged bsa first before the nano particle was to make a standard against which to compare with the Aunp+dye fluorescence to eventually determine concentration whether or not tagging occured

Notes

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