Biotech lab 5 Flashcards
After adding ammonium bicarbonate (50mM) to 10 mL of ddH2O, what pH should be observed?
8-9
What is in the destaining solution?
1 mL of acetonitrile and 1 mL of 50 mM ammonium bicarbonate
What band is taken from the SDS-PAGE gel?
the PTEN band, minimize gel amount and use clean scalpel
What is added to the gel band?
destain solution
Once the destain solution is added to the gel band, what is done?
bench top incubator at 37C for 15 minutes until band becomes clear
How is the gel dehydrated?
adding 300uL of acetonitrile
what does acetonitrile do?
reduce hydrophobic interactions of CBB and gel
after wash with acetonitrile, where is the gel piece put?
speed vacuum for 20 minutes
what protease is used?
trypsin
what is in the trypsin digestion buffer?
resuspended trypsin, ammonium bicarbonate
how is the sample rehydrated?
adding enout trypsin digestion buffer to cover the gel piece
the gel and digestion buffer is incubated where?
on ice for 10 mins
how will you know that the trypsin has entered the matrix?
gel piece should swell
why is parafilm added after the on ice incubation?
to minimize evaporation
where is the gel sample incubated after adding parafilm?
bench top shaking incubator for 2 hrs at 37C
if gel isnt covered, what do you add?
ammonium bicarbonate
How often do you check the gel?
every 30 minutes
after the incubation, what is added for peptide extraction?
vortex and spin down, add formic acid solution and collect supernatant
After collecting supernatant what do you do?
add 1 ul of sample in maldi pate and add 1 ul of UV absorbing maldi matrix
