Module 2 Flashcards
western blot analysis
- ## quantifyy protein expression
what are the steps involved in western blot analysis
- isolate proteins from the sample
- quantifyy the proteins by identifying units of the protein
- load equal concentrations from each sample in a gel matrixx and separate by size
- transfer the separated ones onto a membranee
- block the membrane
- primary antibody to detect protein of interest
- secondary antibody
- add substrate
how to isolate intracellular proteins in western blot analysis
rupture cells to release proteins using detergent solution
how to isolate extract;;I;ar proteins in western blot analysis
grind the tissue using a homogenizer (no detegernt needed)
why transfer from gel to a membrane in western blot analysis
membrane is a more resilient structure
why blocking?
what is special about the secondary antibody in western blot analysis
it contains an enzyme tag
- when chemiluscent substrate is added, it allows for teh visualization of th protein of interest
what is the importance of protein quantification
was protein isolation equivalent for the two samples being compared
- done in mg/mL
what is an important part of protein isolation
keep the proteins cold; proteases dont function well in cold temperatures, protects teh samples
