User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2015/10/06

Biomaterials Design Lab

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Objective Roberta, Maxi, and Asya are running an experiment on the Ocean Optics spectrometer today. I will advise you of some changes to the procedure. Nouf, Loukas, and Melvin and Sydney, Andrew, and Matt will measure protease kinetics with the Bradford Assay using 1uM of protease. I will advise you of slight changes to the procedure. Jamie, Nicole, and Benjamin and Michael and Becky will measure protease kinetics with the Fluorescence Assay using 1uM of protease. I will advise you of slight changes to the procedure. I think that we've been centrifuging our samples at too fast a speed. I checked through Paul and Monika's notebooks. They centrifuged their samples at 300 rpm. I think that, by spinning our samples so fast, we may be compromising them for our other measurements. So, I want to try going a little slower. I made new samples last Friday. Bradford Analysis of Protease Degradation Note: You will be making samples (and blanks) for analysis at different points of time. So plan wisely. All samples will be incubated in eppendorf tubes, which will be placed in the 37C 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). Protease Prep Place the 1mL of Tris/CaCl2 buffer in the pre-measured protease sample and record the concentration Sample Prep To a fiber sample tube (that has been centrifuge and had the supernatant removed) add the appropriate amounts of buffer and protease The total volume should be 1mL The final protease concentration should be 1uM Vortex the sample to disperse the fibers Blank Prep (you will have one blank to match each sample) To a clean eppendorf tube add the appropriate amounts of buffer and protease The total volume should be 1mL The final protease concentration should be 1uM Incubate Sample and Blanks Place the tubes (for both the Blanks and Samples) in the shaker with the 37C water bath Measurement Remove the tubes (the sample and a corresponding blank) from the water bath at the appropriate time For the case of a sample with fibers, centrifuge the sample for 1 min to pull any fibers to the bottom of the tube To a plastic cuvette add 600 uL of pre-mixed Bradford dilution (that I give you) 750 uL of sample 1650 uL of buffer Record the UV-Vis spectrum from 400-800 nm 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) Note: You will be making samples (and blanks) for analysis at different points of time. So plan wisely. All samples will be incubated in eppendorf tubes, which will be placed in the 37C 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). Protease Prep Place the 1mL of phosphate buffer in the pre-measured protease sample and record the concentration Sample Prep To a fiber sample tube (that has been centrifuged and had the supernatant removed) add the appropriate amounts of buffer and protease The total volume should be 1mL The final protease concentration should be 1uM Vortex the sample to disperse the fibers Blank Prep (you will have one blank to match each sample) To a clean eppendorf tube add the appropriate amounts of buffer and protease The total volume should be 1mL The final protease concentration should be 1uM Incubate Sample and Blanks Place the tubes (for both the Blanks and Samples) in the shaker with the 37C water bath Measurement Remove the tubes (the sample and a corresponding blank) from the water bath at the appropriate time For the case of a sample with fibers, centrifuge the sample for 1 min to pull any fibers to the bottom of the tube Blank measurement From the blank measurement, take 20uL of blank and place in a 600uL eppendorf tube Add 140uL of Assay Buffer Add 40uL of Assay Reagent Take Measurement Sample Measurement From the sample, take 20uL of sample and place in a 600uL eppendorf tube Add 140uL of Assay Buffer Add 40uL of Assay Reagent Take Measurement Excitation at 390 nm Emission from 400 to 650 nm Description Add experimental record here. Include what, how, and why... 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.