User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2015/09/29: Difference between revisions

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# Protease Prep
# Protease Prep
## Place the 1mL of phosphate/CaCl2 buffer in the pre-measured protease sample and record the concentration
## Place the 1mL of Tris/CaCl2 buffer in the pre-measured protease sample and record the concentration
# Sample Prep
# Sample Prep
## To a dried fiber sample tube (from one of your previous fiber syntheses in the eppendorf tubes) add the appropriate amounts of buffer and protease
## To a dried fiber sample tube (from one of your previous fiber syntheses in the eppendorf tubes) add the appropriate amounts of buffer and protease

Revision as of 10:33, 30 September 2015

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Objective

Three sets of activities today. Everyone will be working on protease experiments.

  1. Loukas, Melvin, and Nouf will be using the ocean optics spectrometer and working with a protease concentration of 1uM.
  2. Sydney, Andrew, Matt, Roberta, Maxi, and Asya will be doing a Bradford analysis on protease degraded samples.
  3. Jamie, Nicole, Benjamin, Michael, and Becky will be making standardized curves for determining peptide concentration using fluorescence analysis

Ocean Optics

Use your previous protocol for this

Bradford Analysis of Protease Degradation

Note: You will be making samples for analysis at different points of time. So plan wisely.

All samples will be incubated in eppendorf tubes, which will be placed in the 35C water bath on the shaker. Samples should all contain the same concentration of materials.

You should make samples for measurements at 10 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hr, 1.5 hr, 2 hr. (Note, this is for the first time you do this. As you get better, I will expect you to get more measurements done).

  1. Protease Prep
    1. Place the 1mL of Tris/CaCl2 buffer in the pre-measured protease sample and record the concentration
  2. Sample Prep
    1. To a dried fiber sample tube (from one of your previous fiber syntheses in the eppendorf tubes) add the appropriate amounts of buffer and protease
      1. The total volume should be 1mL
      2. The final protease concentration should be 1uM
      3. Vortex the sample to disperse the fibers
  3. Blank Prep (you will have one blank to match each sample)
    1. To a clean eppendorf tube add the appropriate amounts of buffer and protease
      1. The total volume should be 1mL
      2. The final protease concentration should be 1uM
  4. Incubate Sample and Blanks
    1. Place the tubes in the shaker with the 37C water bath
  5. Measurement
    1. Remove the tubes (the sample and a corresponding blank) from the water bath at the appropriate time
    2. For the case of a sample with fibers, centrifuge the sample for 1 min to pull any fibers to the bottom of the tube
    3. To a plastic cuvette add
      1. 600 uL of pre-mixed Bradford dilution (that I give you)
      2. 750 uL of sample
      3. 1650 uL of buffer
    4. Record the UV-Vis spectrum from 400-800 nm
  6. Using the Bradford standardization curve and your data from the 0 fiber sample, determine the concentration of released protein in your solution.

Fluorescence Analysis of Protease Degradation

We are using the Pierce quantitative fluorometric peptide assay. (product link here)

  1. Perform a protease digest of lysozyme
    1. Make a stock lysozyme solution (~ 1mg/mL) using phosphate buffer
    2. Make your protease stock by adding 1mL to your pre-measured protease
    3. Make a sample that has a final volume of 1mL and protease concentration of 1uM
      1. Add the appropriate volume of protease sample to a clean eppendorf tube
      2. Add a volume of lysozyme solution so that the final volume is 1mL
    4. Make a blank sample (no lysozyme)
      1. Add the appropriate volume of protease sample to a clean eppendorf tube
      2. Add a volume of buffer solution so that the final volume is 1mL
    5. Add both tubes to the 37C water bath for 1hr
    6. Make standards for analysis
      1. Standard A: 150uL of reaction sample
      2. Standard B: 75uL of Standard A and 75uL of water
      3. Standard C: 75uL of Standard B and 75uL of water
      4. Standard D: 75uL of Standard C and 75uL of water
      5. Standard E: 75uL of Standard D and 75uL of water
      6. Standard F: 75uL of Standard E and 75uL of water
      7. Standard G: 75uL of Standard F and 75uL of water
      8. Standard H: 75uL of Standard G and 75uL of water
    7. Make blanks for analysis
      1. Blank A: 150uL of blank sample
      2. Blank B: 75uL of Blank A and 75uL of water
      3. Blank C: 75uL of Blank B and 75uL of water
      4. Blank D: 75uL of Blank C and 75uL of water
      5. Blank E: 75uL of Blank D and 75uL of water
      6. Blank F: 75uL of Blank E and 75uL of water
      7. Blank G: 75uL of Blank F and 75uL of water
      8. Blank H: 75uL of Blank G and 75uL of water
  2. Make Samples for Measurement
    1. For each of the standards and blanks described above mix the following
      1. 20uL of sample
      2. 140uL of Assay Buffer
      3. 40uL of Assay Reagent
  3. Take Measurement
    1. Excitation at 390 nm
    2. Emission from 400 to 650 nm

Data

  • Add data and results here...

Notes

This area is for any observations or conclusions that you would like to note.


Use categories like tags. Change the "Course" category to the one corresponding to your course. The "Miscellaneous" tag can be used for particular experiments, as instructed by your professor. Please be sure to change or delete this tag as required so that the categories remain well organized.




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