Second Edition, Version 2.03
April 2017
PlasmaDive
™ Mini Manual, © Biognosys AG, Switzerland
Page 11 of 22
B. Dilution
14. Prepare 1x Dilution Buffer by mixing in a separate 15 ml tube
1 ml of 10x Dilution Buffer and 9 ml of water.
15. Using a single channel pipette, add 150 µl/tube of 1x Dilution
Buffer to a
new
set
of 1.5 ml tubes.
16. Using a single channel pipette, add 15 µl/tube of denatured
plasma sample (from Section A, step 13).
Optional: you might freeze remaining sample in the tubes for a repeated
analysis.
17. Check pH to be 8
–9 in a few samples by pipetting 0.5 µl onto a
pH paper or by using a pH-electrode (Note 8).
17.1. If pH is below 8, adjust it in
all
samples using 5 µl of
10x Dilution Buffer.
17.2. Check again in a few wells, repeat steps 17.1 and 17.2 if
necessary.
C. Digestion using endoprotease trypsin
18. Thaw trypsin (0.4 µg/µl) and spin down briefly.
19. Add 5 µl/tube of trypsin to the tubes with samples from Section
B using a single channel pipette.
20. Gently shake the samples on thermomixer for 1 min at room
temperature.
21. Briefly spin down to collect all liquid at the bottom of the tubes.
22. Incubate the samples at +37°C, 600 rpm in the thermomixer
for 3 hours.
23. Acidify samples by adding 20 µl/sample of 10% (v/v) TFA
solution using a single channel pipette (Note 9).
24. Check pH to be below 2 in a few samples by pipetting 0.5 µl
onto a pH paper or by using a pH-electrode.
24.1. If pH is above 2, adjust it in
all
samples adding 5 µl of
10% (v/v) TFA solution.
24.2. Check again in a few samples, repeat steps 24.1 and 24.2
if necessary.
25. Gently shake the samples on thermomixer for 1 min at room
temperature.
26. Briefly spin down to collect all liquid at the bottom of the tubes.
Optional: if convenient store samples at -20°C until C18 clean-up.
D. Sample clean-up using MicroSpin columns
Spin columns preparation
27. Place 20 MicroSpin columns in a
new
set
of 2 ml centrifuge
tubes.
Note 8
Avoid sample cross-
contamination by using
fresh tips or cleaning
the electrode with water
for every sample.
Note 9
Foaming is possible;
add 10% (v/v) TFA
solution slowly.