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

Biomaterials Design Lab

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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 13.6µM 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 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 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.