Reagents that need to be made fresh before starting:
50mM ammonium bicarbonate in 6M urea
To make a 10ml solution
For ammonium bicarbonate (NH4HCO3)-must use within 24hrs of making it.
(79.06g/mol)x(1mol/1,000mmol)x(50mM/1L)x(1L/1,000ml)x(1,000mg/1g)x 10ml = 39.53 mg of NH4HCO3 for 10ml
Dissolved 39.53mg NH4HCO3 into 5ml nanopure.
(60.06g/mol)x(6mol/L)x(1L/1000ml)x 5ml= 3.6g Urea to add to the 5ml.
Dissolved 3.6g Urea into the solution you just made. Topped off with nanopure to 10ml.
To make a 25ml solution (800ul required/sample)-Must use within 24hrs of making it.
(79.06g/mol)x(1mol/1,000mmol)x(25mM/1L)x(1L/1,000ml)x(1000mg/1g)x 25ml = 49.4mg of NH4HCO3 for 25ml
Dissolved 49.4mg NH4HCO3 in 20ml nanopure by vortexing. Then topped off with nanopure to 25ml.
1) Set up heating block and warmed up to 37C. Verified temperature with second thermometer.
2) Got samples out of -80C as well as one aliquot of previously-made TCEP.
3) Vortexed samples gently and for each sample I pipetted the equivalent of 100ug of protein to a new labeled snaptop centrifuge tube. The protein concentrations were determined by the BCA assay completed on 11/23/2016. I put the leftover samples back in the -80C.
4) I diluted each sample to 100ul using the 50mM NH4HCO3 in 6M urea. Vortexed.
5) I added 6.6ul of 1.5M Tris pH 8.8 to each sample.
6) Then I added 2.5ul of 200mM TCEP and vortexed.
7) The pH was checked by adding 2ul from each sample to a pH test strip and verifying that the pH was over 7.0. I added the drop to the green square as the color will turn bluish if over pH7-8.
8) Then I put the 22 samples in the 37C heating block for 1 hr.
9) I got 3-200ul IAA aliquots from the -80C and wrapped the tubes in foil to keep out of the light.
10) Defrosted and added 20ul of IAA to each sample.
11) Incubated for 1 hr in the dark at room temp.
12) Defrosted 3- 200ul DTT aliquots and added 20ul to each sample.
13) Incubated for 1 hr at room temp.
14) Added 450ul nanopure water to the 2AU Lys-C bottle which will create an enzyme solution of 2ug/ml. I added 1.65ul to each sample which is a 1:30 ratio of enzyme to protein.
15) Incubated for 1 hour at room temp.
16) Added 800ul 25mM NH4HCO3 and 200ul HPLC grade methanol.
17) Got 4 trypsin bottles each with 20ug of trypsin out of the freezer. Added 20ul of nanopure to each of the four bottles. The concentration is 1ug/ul so I will add 3.3ul to each sample for a 1:30 enzyme to protein ratio.
18) Incubated samples overnight.
19) Evaporated samples at 4C in the Genome Sciences speed vac (took about 10 hours).