User:Monika Gasiorek/Notebook/CHEM-571 2014F/2014/09/23

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September 23, 2014

SDS-PAGE Electrophoresis

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.

  • 200 mL of the 10x buffer was added to a 2000 mL volumetric flask

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:

  • The samples were heated for 5 min at 100C in the thermocycler pre-loading

Well Sample
1 Ladder
3 1g/1L BSA stock
5 ___:__ [Au]:[BSA] colloid
7 1g/1L Lysozyme stock solution
9 36.5:1 [Au]:[Lys] colloid
11 1g/1L soy stock solution

III. A 12 well pre-cast Mini Protean TGX gel was obtained and prepared.

  • The comb and tape was removed from the gel
  • The wells of the gel were rinsed with the prepared running buffer dilution

IV. The Bio-Rad Mini Protean system electrophoresis cell was assembled.

  • The inner and outer buffer chambers were filled with the diluted running buffer to the "4 gel" mark.

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

  • this step was repeated with fresh destain solution 2 more times

Bradford Assay - Take 2

I. A 0.5 g/L lysozyme stock solution was prepared.

  • 0. 50350 g of solid lysozyme was dissolved in 1 L of 0.90378% saline solution
    • the saline solution was prepared by dissolving 9.0378 g of NaCl in a 1000 mL volumetric flask

II. 8 serial dilutions were done to prepare 9 5 mL samples of lysozyme stock solution of varying concentrations.

  • The dilutions were done using 5 mL of each preceding dilution, to which 5 mL of a 50 mM NaCl/Tris solution was added, halving the concentration of lysozyme with each dilution

The following chart demonstrates the preparation of each UV-Vis sample consisting of 800 μL of each dilution and 200 μL of Bradford reagent.

Sample ' [Initial Lysozyme] (μg/mL) Amount of Sample (μL) Amount of Bradford reagent (μL) [Final Lysozyme] (μg/mL)
1503.500800200402.800
2251.750800200201.400
3125.875800200100.700
462.93880020050.350
531.46980020025.175
615.73480020012.588
77.8678002006.294
83.9348002003.147
91.9678002001.573

III. Two UV-Vis blanks were run.

  • one of the 50 mM tris solution, the other of a 50 mM tris and Bradford reagent solution mixture

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.

10/1/2014

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.

Image:Bradford_Analysis_Take_2_Corrected.png

Image:Bradford_Analysis_Take_2_Corrected_Curve.png



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