User:Zachary I. Mendel/Notebook/Zacks Notebook/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)
- Note: This is where coordination is a good thing. Take 1 spectrum at a time. Let other people go.
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)
Making buffer: 1L 50mM Tris 50mM NaCl pH 7.5
Should mass out:
Gold solution for group: HAT to make nano particles
Today's experiment certainly did not go as planned. We took absorbance readings for three trials of the adenosine deaminase and did not get good readings on any of the trials. Dr. Hartings has instructed us to repeat the experiment tomorrow the 11th using 2ml volumes as opposed to 1ml readings. He hopes this will correct the problem.
It is important to note that when we were making our initial stock solution of adenosine deaminase, we were only able to obtain 3.67ug of protein as opposed to the 10ug that we were hoping to obtain.