Practical 2 Flashcards
What technique was used first to identify mw of the glut 4 in each sample
Sds page
How would you get cytoplasmic and plasma membrane fractions of glut 4 from adipocytes
Centrifugstuon of the wt and mutant adipocytes
Why wouldn’t glut4 be seen in membrane with adipocytes mutant
Loss of pkc signalling from insulin means glut 4 can’t travel to membrane
Which sample will have the lowest intensity of protein in western blot
Cytoplasmic wt with insulin - all the glut 4 goes to membrane
What is in the sds loading gel x 10
Glycerol Sds Dtt EDTA metal chelator Dye
What further denatures proteins in sds
Heat
What type of membrane was the gel pressed onto via western blot
Nitrocellulose
What type of buffer was used to add each layer on western blot
Transfer buffer
What is blocking buffer/agent
Something with milk powder or casein in it , it fills the empty protein binding spaces on the membrane so pa and sa don’t non specifically bind
As well as casein what was in blocking buffer
Tbst (with tween 20)
What was wash buffer with tbst tween 20 for
Wash non specific protein binding from the membrane
Which enzyme was the sa conjugated with
Alkaline phosphatase
What was the developer solution
The chromogenic substrate added to alkaline phosphatase
What is the product called between the alkaline phosphatase and the chromogenic substrate picked up on western blot
Chemiluminescent product
What would more than 1 band on western blot indicate
Non specific binding of ab to another protein on membrane
