User:Trisha I. Ibeh/Notebook/Trisha Notebook/2013/09/10: Difference between revisions

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The basic protocol can be found [http://www.bio-rad.com/webroot/web/pdf/lsr/literature/LIT33.pdf here]. (*Note: use section 2.3, page 5)
The basic protocol can be found [http://www.bio-rad.com/webroot/web/pdf/lsr/literature/LIT33.pdf here]. (*Note: use section 2.3, page 5)


# Make a stock solution that is roughly 10mg protein in 2mL of buffer. (there are 2-2mL volumetric flasks that you will have to share)
# Make a stock solution that is roughly 9.9mg protein in 2mL of buffer. (there are 2-2mL volumetric flasks that you will have to share)
# Calculate what your actual concentration
# Calculate what your actual concentration
# Make 6 standard solutions (1mL each) between 1μg/mL and 10μg/mL
# Make 6 standard solutions (1mL each) between 1μg/mL and 10μg/mL

Revision as of 04:23, 5 October 2013

Biomaterials Design Lab <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
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The template for this lab can be seen from Dr. Hartings lab. Values are altered to accurately describe the lab that was conducted on this day. The template can be found here

Objective

Make a calibration curve for different proteins that will be used during the semester with the Bradford Assay.The protein of choice was bovine serum albumin (BSA)and using the procedure provided above, 6 different standards were created and analyzed using UV- Vis.

Procedure

The basic protocol can be found here. (*Note: use section 2.3, page 5)

  1. Make a stock solution that is roughly 9.9mg protein in 2mL of buffer. (there are 2-2mL volumetric flasks that you will have to share)
  2. Calculate what your actual concentration
  3. Make 6 standard solutions (1mL each) between 1μg/mL and 10μg/mL
    1. Determine the appropriate volume of your stock to use (for the proper final concentration in 1mL) and add that volume to an eppendorf tube
    2. Add 200μL of the Bio-Rad Protein Assay reagent
    3. Add the correct amount of buffer such that the final volume is 1mL
    4. Close the tubes and vortex them.
    5. Let them sit for 5 minutes
  4. Take a UV-Vis (no less than 1 hour after they were produced).
    1. Use the plastic cuvettes.
  5. Make 2 blanks as well (800uL buffer and 200uL Assay reagent) and take it's UV spectrum. (400nm-800nm)
    1. 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)
  6. 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.

  1. Make a calibration curve.
  2. Determine if you need to redo any data or sample prep.

The Atomic Absorption standards will be made for the following day.

Using the gold AA/ICPMS standard solution make 5 new solutions (Note: Use water - NOT BUFFER - to make these solutions)

  1. 25 ug/mL
  2. 20 ug/mL
  3. 15 ug/mL
  4. 10 ug/mL
  5. 5 ug/mL

Data


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