User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2013/09/10: Difference between revisions
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## Use the plastic cuvettes. | ## Use the plastic cuvettes. | ||
# Make a blank as well (800uL buffer and 200uL Assay reagent) and take it's UV spectrum. | # Make a blank as well (800uL buffer and 200uL Assay reagent) and take it's UV spectrum. | ||
# After you have finished one set, repeat the process (make new samples and new measurements) | |||
- Note: This is where coordination is a good thing. Take 1 spectrum at a time. Let other people go. | |||
# Make a calibration curve. | |||
# Determine if you need to redo any data or sample prep. | |||
==Data== | ==Data== |
Revision as of 08:06, 6 September 2013
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ObjectiveThe 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. DescriptionThe 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.
Data
NotesThis area is for any observations or conclusions that you would like to note.
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