This method can help answer key questions in the nanoscience field, such as fluorophore formation and middle protein complexes. The main advantage of this technique is that it helps to illustrate specific metal binding sites and proteins. First, dissolve 25 milligrams of BSA in one milliliter of HPLC grade water in a five milliliter reaction vial containing a magnetic stir bar.
Next, prepare a five millimolar solution of chloroauric acid and HPLC grade water. Place the reaction vial containing the BSA solution in a 37 degree Celsius water bath and stir at 750 RPM using a magnetic stirrer. Immediately after the stirring begins, add one milliliter of the chloroauric acid solution to the BSA solution.
After stirring for two minutes, add 100 microliters of one molar sodium hydroxide to the solution to bring the PH to 12. Dissolve 25 milligrams of BSA in one milliliter of HPLC grade water in a reaction vial. Next, prepare a five millimolar solution of copper chloride dihydrate in HPLC grade water.
Add one milliliter of the BSA solution to a five milliliter reaction vial containing a magnetic stir bar, and place it in a water bath at 37 degrees Celsius. Stir the solution at 750 RPM. Immediately add 0.5 milliliters of the copper chloride dihydrate solution to the reaction vial.
After stirring for two minutes, add 75 microliters of one molar sodium hydroxide to the solution to bring the PH to 12 and stir for two hours. Following this, prepare a five millimolar solution of chloroauric acid in HPLC grade water. Add 0.5 milliliters of the chloroauric acid solution to the reaction vial.
Then, adjust the PH to 12 using one molar sodium hydroxide and stir the solution for two hours. Add one milliliter of BSA solution to a five milliliter reaction vial containing a magnetic stir bar and place it in a water bath at 37 degrees Celsius. Stir the solution at 750 RPM.
Immediately add 0.5 milliliters of a previously prepared five millimolar nickel chloride hexahydrate solution to the reaction vial. After stirring for two minutes, add 75 microliters of one molar sodium hydroxide to bring the PH to 12 and stir for two hours. Add 0.5 milliliters of five millimolar chloroauric acid solution to the reaction vial.
Then, adjust the PH to 12 using one molar sodium hydroxide and stir the reaction mixture for two hours. Dissolve two milligrams of N-Ethylmaleimide in one milliliter of PBS. Then, dissolve two milligrams of BSA in one milliliter of the maleimide solution.
Transfer the solution to a five milliliter reaction vial containing a magnetic stir bar and stir at 20 degree Celsius and 500 RPM for one hour. After one hour, dialyze the solution using 12 kilodalton dialysis tubing in 500 milliliters of PBS. Stirring at 50 RPM with a magnetic stirrer overnight to remove unreacted and ethylmaleimide.
On the following day, transfer the reaction vial containing the maleimide solution to a water bath at 37 degrees Celsius and stir at 750 RPM. Immediately, add a previously prepared 0.4 millimolar chloroauric acid solution to the reaction vial. After stirring for two minutes, add 75 microliters of one molar sodium hydroxide to the solution to bring the PH to 12 and stir for two hours.
Prepare a solution of two molar urea and 50 millimolar ammonium bicarbonate in HPLC grade water. Then, dissolve 3.3 milligrams of BSA in one milliliter of the urea solution and transfer to a five milliliter reaction vial containing a magnetic stir bar. Next, prepare a stock solution of 0.25 molar TCEP by dissolving 62.5 milligrams of TCEP and one milliliter of HPLC grade water.
Add the stock solution to the reaction vial until the final concentration of TCEP is eight millimolar. Then, incubate the solution in a water bath for one hour at 50 degree Celsius with stirring at 500 RPM. In the meantime, prepare a 100 nanomolar stock solution of N-ethylmaleimide by dissolving 12.5 milligrams of N-ethylmaleimide in one milliliter of HPLC grade water.
Add the stock solution to the cooled reaction vial until the final concentration of N-ethylmaleimide is 16 millimolar. Then, stir at 20 degrees Celsius and 500 RPM for two hours. Dialyze the solution using 12 kelodalton dialysis tubing in 500 milliliter of 50 millimolar ammonium bicarbonate, stirring at 50 RPM with a magnetic stirrer overnight to remove excess reagents.
On the following day, transfer the reaction vial to a water bath at 37 degrees Celsius and stir at 750 RPM. Immediately, add one milliliter of previously prepared 0.66 millimolar chloroauric acid solution to the reaction vial. After stirring for two minutes, add one molar sodium hydroxide to the solution until the PH is 12 and stir for two hours.
From the fluorescence of BSA gold three complex, it has been observed that the conversion of the intrinsic blue fluorescence of BSA to red fluorescence occurs at about PH 9.7 through an equilibrium transition. Excitation-emission maps of BSA gold three at different BSA to gold molar ratios show how altering the molar ratios yields the same emission wavelength at different excitation wavelengths. Copper, Nickel, and Gold competitively bind to a known site in BSA.
The cysteine 34 capped BSA shows a change in excitation-emission map peak patterns upon gold binding and these results show how altering of specific binding sites alters fluorescence patterns. The all-thiol-capped-BSA shows no red fluorescence and reveals cysteine-cysteine disulfide bonds as possible binding sites to produce the red fluorophore. While attempting this procedure, it is important to remember to adjust the PH as described or to use the correct buffer because fluorophore formation is dependent on the PH-induced change of protein confirmations.
