User:Nancy T. Miles/Notebook/N.Miles Lab 2013-09-03/2013/09/10

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Objective

To use protein assay (The Bradford Method) to measure the amount of hemoglobin in 6 stock solutions using UV-Vis Spectroscopy

Methods

Standard Solution Concentrations (μg/mL) Volume of Stock solution (mL) Volume of TRIS Buffer (mL)
1 0.0002 0.7998
2 0.0004 0.7996
4 0.0008 0.7992
5 0.001 0.799
8 0.0016 0.7984
9 0.0018 0.7982 Blank 0 0.800
  • 200μL of the protein Assay reagent was added to each standard solution to bring the total volume of each sample to 1 mL.
  • p2 and p1000 micro-pipettes were used for these volumes.

Atomic Absorption Measurement Standards The following 5 samples of diluted gold solutions were made with water: 25μg/mL, 20μg/mL, 15μg/mL, 10μg/mL, and 5μg/mL.

  • 10 mL of each of these solutions were made.

Data

The following graphs represent the UV-Vis absorbance specta of hemoglobin. The procedure was run twice. Image:20130910_hemogAbs.png

  • The following tables specify the absorbances of each concentration of solution at 600 nm.
Standard Solution Concentrations (μg/mL) Absorbances; Set 1 Standard Solution Concentrations (μg/mL) Absorbances; Set 2
1 0.104 1 0.157
2 0.165 2 0.219
4 0.294 4 0.266
5 0.325 5 0.355
8 0.401 8 0.41
9 0.435 9 0.445

The following graph represents the calibration curve of the absorbances of hemoglobin from both sets of data.

Image:20130910_hemogCali.png



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