User:Daniel-Mario Larco/Notebook/AU Biodesign Lab - 09/03/2013/2013/09/10
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The primary way of determining protein concentration is through a measurement of the protein's UV-Vis spectrum and using its molar absorptivity at 280nm to calculate concentration. For low concentrations of proteins, UV-Vis of just the protein is often not sensitive enough to accurately measure concentration. During the semester, we may need to measure protein concentrations that are very low. One chemical tool that we can use to do this is called the Bradford Assay. The Bradford Assay makes use of the Coomassie Blue dye, which binds to proteins. Upon binding to a protein, this dye undergoes a change in its absorption features. (No protein: peak at 460. Protein: peak at around 600). We will be making calibration curves (using the Bradford Assay) for the different proteins we'll be using throughout the semester.
The basic protocol that we will be using for this procedure can be found here. (*Note: use section 2.3, page 5)
* The concentration of the stock solutions were 5.6μg/mL for the first trial and 5.9μg/mL for the second.
- Because the absorbance of the 2.5μg/mL and 3.0μg/mL were above 1,we made two other samples of 0.25μg/mL and 0.1μg/mL and reran them and got a new calibration curve.
Note - Solutions from today containing the stain will go into a waste container in the hood.
We are also going to make Atomic Absorption standards for tomorrow!
Using the gold AA/ICPMS standard solution make 5 new solutions (Note: Use water - NOT BUFFER - to make these solutions)
- The standard solutions obtained were put in plastic tubes and saved for the next lab. THey were made by diluting the gold AA/ICPMS standard solution that had a concentration of 1000 ± 10 μg/mL. It was diluted with distilled water.