- Begin the new-synthesis method using 78% BSA-Cys by mass. We had to switch to percentage by mass because molar percentage required too much cysteine.
- Continue Data analysis (UV/VIS, Fluorescence) from the samples synthesized on 2/26/14
This protocol was taken from Matt Harting's notebook
- To a 3-necked flask is added
- 25mL of 20 mM L-cysteine (roughly 22% L-Cysteine)
- 25mL .1459 mM BSA (roughly 78% by mass)
- 5mL of 100mM ZnSO4 (0.5 mmol)
- 5.437mL of 10.3mM MnCl2 (0.056 mmol)
- The pH of this mixture is adjusted to 11 with ~1M NaOH (should be on the order of 60uL)
- This mixture is stirred under Ar (I'll use N2) for 30 minutes
- 5.505mL of 101mM Na2S is quickly added.
- The solution is stirred for 20 minutes
- The solution is then aged at 50C under air for 2 hours.
Absorbance 80% BSA-L-cysteine double concentration
Fluorescence 80% BSA-L-cysteine double concentration
Absorbance 80% BSA-L-cyisteine (large volume: ~20mL)
Fluorescence 80% BSA-L-cysteine (large volume: ~20mL)
- In the past we used molar ratios to to relate the ligand to protein ratios, for this specific synthesis method that would have used too large an amount of BSA (approximately 330g) so we instead have started using mass ratios, and this synthesis is the first that we are going to be using it for.