User:Monika Gasiorek/Notebook/CHEM-571 2014F/2014/09/23
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September 23, 2014
I. A 10x SDS-PAGE Running Buffer (30.3 g Tris, 144.1g Clycine, 10g SDS, water to 1L) was diluted by a factor of 10.
II. 10-20 μL of each of the samples prepared 9/17, in addition to a ladder/marker, was loaded into one of six wells in the following order:
III. A 12 well pre-cast Mini Protean TGX gel was obtained and prepared.
IV. The Bio-Rad Mini Protean system electrophoresis cell was assembled.
V. The gel was run for approximately 30 min at 200 V, 0.05 A, and 10 W.
VI. After 30 min, the gel was placed in a Fixative Solution (40% methanol, 10% acetic acid, 50% water) for 30 minutes
VII. The gel was then placed in a Stain Solution (0.025% (w/v) Coomassie Blue, 10% acetic acid, 90% water) for 1 hour
VIII.Finally, the gel was placed in Destain Solution (10% acetic acid, 90% water) for 15 minutes
Bradford Assay - Take 2
I. A 0.5 g/L lysozyme stock solution was prepared.
II. 8 serial dilutions were done to prepare 9 5 mL samples of lysozyme stock solution of varying concentrations.
The following chart demonstrates the preparation of each UV-Vis sample consisting of 800 μL of each dilution and 200 μL of Bradford reagent.
III. Two UV-Vis blanks were run.
IV. Consequently, a UV-Vis spectrum was obtained for each of the 9 prepared samples, demonstrating the following data for a Beer's Plot calibration curve:
NOTE:The results at first were very similar to those obtained two weeks ago, rather inconclusive with a lack of linearity. However, the concentrations of the samples were well above what was hypothesized to work for this process. The values obtained for concentrations between 1 and 20 μg/mL demonstrated a linear result with R^2 = 0.986. However, this experiment could be repeated again with a larger number of samples with concentrations in the specified concentration range.
The data obtained from the second take of Bradford Analysis performed on 9/23 was corrected today to account for varying baselines values. All data was adjusted so that each spectra began at 0 absorbance for 800 nm.