User:Matt Hartings/Notebook/AU Biomaterials Design Lab/2015/10/06: Difference between revisions
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## Place the 1mL of Tris/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 | ## 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 total volume should be 1mL | ||
### The final protease concentration should be 1uM | ### The final protease concentration should be 1uM | ||
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## Record the UV-Vis spectrum from 400-800 nm | ## 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. | # 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 [https://www.thermofisher.com/order/catalog/product/23290 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 | |||
Revision as of 17:58, 5 October 2015
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Objective
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 DegradationNote: 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).
Fluorescence Analysis of Protease DegradationWe 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).
Description
Data
NotesThis area is for any observations or conclusions that you would like to note.
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