Flashcards in Biotech-western blotting and chemiluminescence Deck (20)
Once the membrane is taken out of the fridge, it must be reactivated. How is this done?
Brief (30 second) incubation period in methanol.
How is the methanol rinsed off?
By incubating the activated membrane in water.
What is the blocking solution?
TBST buffer with 5% skim milk.
What is the point of the blocking solution?
Blocking solution works by covering the unoccupied areas of the membrane with a dense layer of molecules. This prevents non-specific binding of primary and secondary antibodies.
Once the blocking buffer is decanted, what is added?
The primary antibody solution.
Once the blocking solution is added, how long is this incubated for?
1 hour on orbital shaker at room temperature
What is the primary antibody solution?
1:2000 dilution of rabbit anti-PEN antibody in 1% skim milk in 1x TBST
How long is the membrane incubated with the primary antibody solution?
For 1 hour with gentle agitation at room temperature
How is the blot that had been incubated with the primary antibody washed?
Washed by decanting the primary antibody solution and adding 30 mL of TBST to the membrane. This is washed for a period of 5 minutes with gentle agitation at room temperature. The wash solution is then decanted and this is repeated 2x.
What is the secondary antibody conjugate solution composed of?
1:5000 dilution of goat anti-rabbit conjugated HRP in 1% skim milk in 1x TBST.
How long is the blot and secondary antibody solution incubated for?
45 minutes using gentle agitation at room temperature.
How is the membrane washed?
decant and add TBST to the membrane, wash and agitate for 5 mins at RT. Repeat twice with additional TBST.
What is done to allow the blot to develop? (after the washing step post-secondary antibody solution)
Remove the membrane and drain the excess liquid without letting the blot dry. Place on a piece of saran wrap on a level surface.
What is added to the 3mL screw cap containing the oxidizing agents?
Enhanced luminol reagent.
What is the ratio of oxidizing agent to luminol?
1:1, 1 mL of each
How is the oxidizing agent and luminol reagent mixed?
Immediately after vortexing, what is done with the solution?
Pipette the substrate solution evenly onto the membrane.
How long is the substrate solution incubated with the membrane for?
How is the membrane removed?
Using tweezers, and draining the excess liquid from the blot.