Flashcards in Biotech-immunoprecipitation Deck (23)
What is used to re-suspend the protein A slurry?
What is pre-clearing?
Preclearing the lysate at the start of each immunoprecipitation experiment is a way to remove potentially reactive components from the cell lysate prior to the IP to prevent the non-specific binding of these components to the IP beads or antibody.
what is done to the precleared lysate once it is added to tubes?
parafilm to prevent sample loss.
At what temperature and for how long are the samples to be IP'd incubated for?
90 minutes at 4C
How are the immuno-precipitated complexes collected after the IP?
Centrifugation at 5000 rpm for 1 minute at room temperature. Take supernatant and leave pellet (which should contain the important stuff)
After IP, and after centrifugation, what does the supernatant contain?
Most likely proteins with non-specific interactions or cell debris.
What does the pellet contain?
hopefully the protein of interest bound to the antibody and bound to the protein a beads.
How are the pellets from IP resuspended?
Using laemmli sample buffer contain beta-mercaptoethanol.
What does beta-mercaptoethanol do?
reduce disulphide bonds (important for SDS, obviously)
What samples are resuspended?
Those before IP and those after IP
After the samples have been resuspended, what else is done?
Heat all samples at 95C for 5 minutes, vortex and spin for 2 minutes.
What is the maximum voltage limit?
What is the max power limit?
What is the maximum ambient temperature limit?
Why are we using chilled (4C) transfer buffer?
improves heat dissipation
how is the PVDF membrane activated?
By soaking in methanol for 30 seconds
Once the membrane is activated, where is it added?
How long is the blot run for and what conditions?
100V for 1 hour
What time interval should the electroblot be monitored%
every 15 minutes
What should the readings be (what should they be adjusted to every 15 minutes)?
reduce or increase voltage to keep current at around 300-350 mA.
How is the membrane removed?
using tweezers and wrap in saran wrap
How are most things cleaned?
with deionized water