I only need help with #2 and #3, I can do the definitions part! Thank you! Readi
ID: 149938 • Letter: I
Question
I only need help with #2 and #3, I can do the definitions part! Thank you!
Reading Questions Week 4 (from posted reading) What are the uses for gel electrophoresis of protein samples? Gel electrophoresis separates macromolecules such as DNA, RNA, and proteins. DNA fragments are separated based on their size, while proteins can be separated by both their size and their charge 1. In an SDS PAGE gel, why do proteins separate by size? SDS-PAGE separates proteins based on their differential rates of migration through a gel under the influence of an applied electrical field. SDS-PAGE primarily separates proteins based on their size because the ionic detergent SDS denatures and binds proteins to make them uniformly negatively charged. So, when a current is applied, all the SDS-bound proteins will migrate through the gel toward the positively charged electrode " Which protein would migrate farther (small or large)? Proteins with less mass travel more quickly through the gel than those with greater mass because of the sieving effect of the gel matrix. 2. In lysed cells, what would be a method to isolate functional mitochondria? 3. How can protein or DNA sequences be used to determine evolutionary pathways? 1. Edman degradation is limited to 50 amino acids. What is the strategy for longer sequences 2. Define the following: a. Antigen b. Antibody c. Polyclonal antibodies d. Hybridoma cell e. ELISA f. Western blottingExplanation / Answer
Answer (2): Mitochondrial Purification Protocol
Reagents needed:
NKM buffer
1 mM Tris HCl, pH 7.4
0.13 M NaCl
5 mM KCl
7.5 mM MgCl2
Homogenization buffer
10 mM Tris-HCl
10 mM KCI
0.15 mM MgCl2
1 mM PMSF
1 mM DTT
Always add PMSF and DTT immediately before use to NKM and homogenization buffer.
Mitochondrial suspension buffer
10 mM Tris HCl, pH 6.7
0.15 mM MgCl2
0.25 mM sucrose
1 mM PMSF
Procedure