User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2013/09/10: Difference between revisions
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# Take a UV-Vis (no less than 1 hour after they were produced). | # Take a UV-Vis (no less than 1 hour after they were produced). | ||
## Use the plastic cuvettes. | ## Use the plastic cuvettes. | ||
# Make | # Make 2 blanks as well (800uL buffer and 200uL Assay reagent) and take it's UV spectrum. (400nm-800nm) | ||
## We will be using one of the blanks as a reference for each spectrum that we take. I'll show you where to place this cuvette for each spectrum you collect) | |||
# After you have finished one set, repeat the process (make new samples and new measurements) | # 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. | - Note: This is where coordination is a good thing. Take 1 spectrum at a time. Let other people go. | ||
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# Determine if you need to redo any data or sample prep. | # Determine if you need to redo any data or sample prep. | ||
Note - Solutions from today will go into a waste container in the hood. | 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! | We are also going to make Atomic Absorption standards for tomorrow! |
Revision as of 08:02, 10 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.
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)
DataMaking buffer: 1L 50mM Tris 50mM NaCl pH 7.5 Should mass out:
Actually measured:
Gold solution for group: HAT to make nano particles
NotesThis area is for any observations or conclusions that you would like to note.
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