User:Paul Rothenberg/Notebook/CHEM 571 Fall 2014/2014/09/23: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 23: Line 23:
The protocol can be found [http://openwetware.org/wiki/User:Matt_Hartings/Notebook/AU_Biomaterials_Design_Lab/2013/09/25 here]. The proteins solutions prepared were placed in 100°C in a thermocycler for 5 minutes to denature the protein. The running buffer was diluted 10X.  
The protocol can be found [http://openwetware.org/wiki/User:Matt_Hartings/Notebook/AU_Biomaterials_Design_Lab/2013/09/25 here]. The proteins solutions prepared were placed in 100°C in a thermocycler for 5 minutes to denature the protein. The running buffer was diluted 10X.  


20 µL of the protein ladder was loaded, along with 20 µL of samples in each well.
20 µL of the protein ladder was loaded, along with 20 µL of samples in each well. The ladder was in well 1, BSA in 3, BSA colloid in 5, Lysozyme in 7, Lysozyme colloid in 9, and Soy Protein in 11.  


The gel was run for 30 minutes at 200V. It was fixed for 30 minutes, stained for 1 hour, and destained for 15 minutes. The destaining step was run three times total.
The gel was run for 30 minutes at 200V. It was fixed for 30 minutes, stained for 1 hour, and destained for 15 minutes. The destaining step was run three times total.


==Redo of Bradford Assay==
==Redo of Bradford Assay==

Revision as of 10:23, 23 September 2014

Project name <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>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

SDS-PAGE of Lysozyme

We are running SDS-PAGE of the samples that Monika prepared. Here is what she did:

5 samples were prepared and stored in the fridge for electrophoresis next week.

  • 20 μL of the 1g/1L lysozyme stock solution were added to a 2 mL volumetric flask. 10 μL of this dilution were added to 10 μL of a running buffer prepared by Dr. Fox
  • 20 μL of the 36.5:1 [Au]:[Lys] colloid solution were added to a 2 mL volumetric flask. 10 μL of this dilution were added to 10 μL of the running buffer
  • 20 μL of a ___:__ [Au]:[BSA] colloid solution created the first day of lab were added to a 2 mL volumetric flask. 10 μL of this dilution were added to 10 μL of the running buffer
  • 20 μL of a 1g/1L BSA stock solution were added to a 2 mL volumetric flask. 10 μL of this dilution were added to 10 μL of the running buffer
  • 0.00209 g of powdered soy protein were dissolved in a 2 mL volumetric flask. 50 μL of this stock soy solution were then added to a 5 mL volumetric flask. 10 μL of this dilution were then added to 10 μL of the running buffer


Procedure for SDS-PAGE

The protocol can be found here. The proteins solutions prepared were placed in 100°C in a thermocycler for 5 minutes to denature the protein. The running buffer was diluted 10X.

20 µL of the protein ladder was loaded, along with 20 µL of samples in each well. The ladder was in well 1, BSA in 3, BSA colloid in 5, Lysozyme in 7, Lysozyme colloid in 9, and Soy Protein in 11.

The gel was run for 30 minutes at 200V. It was fixed for 30 minutes, stained for 1 hour, and destained for 15 minutes. The destaining step was run three times total.

Redo of Bradford Assay

The data was not conclusive for the Bradford assay, so we are re-running it in larger quantities with serial dilutions. 1L of 0.9% saline was prepared, and 0.50350 g of lysozyme was dissolved.




Retrieved from "https://openwetware.org/mediawiki/index.php?title=User:Paul_Rothenberg/Notebook/CHEM_571_Fall_2014/2014/09/23&oldid=822540"