User:Javier Vinals Camallonga/Notebook/Javier Vinals notebook/2013/09/10
|Project name||Main project page|
Previous entry Next entry
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)
- We used pepsin, and we calculated that our actual concentration is 5.6 for trial 1, and 5.9 for trial 2
"Table 1. Concentration and volumes used for the 6 solutions made, Trial 1"
"Table 2. Concentration and volumes used for the 6 solutions made, Trial 2"
"Figure 1. Absorbance vs wavelength in Trial 1 of pepsin"
"Figure 2. Absorbance vs wavelength in Trial 2 of pepsin"
- Note: This is where coordination is a good thing. Take 1 spectrum at a time. Let other people go.
"Figure 3. Calibration curve of Trial 1 pepsin"
"Figure 4. Calibration curve of trial 2 pepsin"
"Figure 5. Calibration curve of both trials"
- We had to redo two samples. We discarded the data of sample 2.5 and 3, because their absorbance was over 1, and we used 0.25 and 0.1 for our calibration curves
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