User:Melvin Colorado Escobar/Notebook/chem 471/2015/09/30: Difference between revisions
From OpenWetWare
(Autocreate 2015/09/30 Entry for User:Melvin_Colorado_Escobar/Notebook/chem_471) |
No edit summary |
||
Line 11: | Line 11: | ||
==Objective== | ==Objective== | ||
* To make standardized curves for determining peptide concentration using fluorescence analysis. | |||
==Description== | ==Description== | ||
* Dr. Hartings left a procedure for us to use today in his lab [[User:Matt_Hartings/Notebook/AU_Biomaterials_Design_Lab/2015/09/29 | notebook]]. | |||
*We are using the Pierce quantitative fluorometric peptide assay. (product link [https://www.thermofisher.com/order/catalog/product/23290 here]) | |||
# Perform a protease digest of lysozyme | |||
## Make a stock lysozyme solution (~ 1mg/mL) using phosphate buffer | |||
## Make your protease stock by adding 1mL to your pre-measured protease | |||
## Make a sample that has a final volume of 1mL and protease concentration of 1uM | |||
### Add the appropriate volume of protease sample to a clean eppendorf tube | |||
### Add a volume of lysozyme solution so that the final volume is 1mL | |||
## Make a blank sample (no lysozyme) | |||
### Add the appropriate volume of protease sample to a clean eppendorf tube | |||
### Add a volume of buffer solution so that the final volume is 1mL | |||
## Add both tubes to the 37C water bath for 1hr | |||
## Make standards for analysis | |||
### Standard A: 150uL of reaction sample | |||
### Standard B: 75uL of Standard A and 75uL of water | |||
### Standard C: 75uL of Standard B and 75uL of water | |||
### Standard D: 75uL of Standard C and 75uL of water | |||
### Standard E: 75uL of Standard D and 75uL of water | |||
### Standard F: 75uL of Standard E and 75uL of water | |||
### Standard G: 75uL of Standard F and 75uL of water | |||
### Standard H: 75uL of Standard G and 75uL of water | |||
## Make blanks for analysis | |||
### Blank A: 150uL of blank sample | |||
### Blank B: 75uL of Blank A and 75uL of water | |||
### Blank C: 75uL of Blank B and 75uL of water | |||
### Blank D: 75uL of Blank C and 75uL of water | |||
### Blank E: 75uL of Blank D and 75uL of water | |||
### Blank F: 75uL of Blank E and 75uL of water | |||
### Blank G: 75uL of Blank F and 75uL of water | |||
### Blank H: 75uL of Blank G and 75uL of water | |||
# Make Samples for Measurement | |||
## For each of the standards and blanks described above mix the following | |||
### 20uL of sample | |||
### 140uL of Assay Buffer | |||
### 40uL of Assay Reagent | |||
# Take Measurement | |||
## Excitation at 390 nm | |||
## Emission from 400 to 650 nm | |||
==Data== | ==Data== |
Revision as of 11:23, 26 November 2015
Biomaterials Design Lab | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
Objective
Description
Data
NotesThis area is for any observations or conclusions that you would like to note.
|