User:Klare Lazor/Notebook/Chem-496-001/2012/02/07

From OpenWetWare

Jump to: navigation, search
Biomaterials Design Lab Main project page
Previous entry      Next entry


Objective

  • making buffer for tagging tom

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 (added extra 2 grams)
    • 137.99g/mol x 0.05 mol/L = 6.89 x .25 L = 1.725 g
    • total solution 250mL


Procedure

  • mixed in 406 microliters of a pH buffer of 7.02, 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
  • used a dialysis bag to separate the free gold and dye from solution

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

Notes

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


Use categories like tags. Change the "Course" category to the one corresponding to your course. The "Miscellaneous" tag can be used for particular experiments, as instructed by your professor. Please be sure to change or delete this tag as required so that the categories remain well organized.


Personal tools