Second Edition, Version 2.03
April 2017
PlasmaDive
™ Mini Manual, © Biognosys AG, Switzerland
Page 12 of 22
28. Add 200 µl of methanol into each column, centrifuge it at
100 x
g
for 1 min and discard flow-through.
29. Add 200 µl/column of C18 Cleaning Solution, centrifuge at
100 x
g
for 1 min, and discard flow-through.
30. Add 200 µl/column of C18 Washing Solution, centrifuge at
100 x
g
for 1 min, and discard flow-through.
31. Repeat step 30 two more times. Make sure the there is no
washing solution left on the column material surface after the
last wash.
Sample loading to spin columns
32. Centrifuge (thawed) samples from Section C at 16000 x
g
for
1 min.
33. Place MicroSpin columns in a
new set
of 2 ml centrifuge
tubes.
34. Load samples (supernatant) into the MicroSpin columns,
centrifuge at 100 x
g
for 1 min,
do not discard flow-through
35. Repeat step 34 by re-loading the flow-through collected in step
34. Discard the flow through (Note 10).
36. Add 200 µl/column of C18 Washing Solution, centrifuge at
100 x
g
for 1 min and discard the flow-through (Note 10).
37. Repeat step 36 two more times.
Elution and sample preparation
38. Place MicroSpin columns in a
new set
of 2 ml centrifuge
tubes.
39. Add 200 µl/column of C18 Elution Buffer, centrifuge at 100 x
g
for 1 min; keep the flow-through in the tubes.
40. Repeat step 39 once again, collecting all eluates in the same
tubes.
Optional: if necessary store the tubes with samples at -20°C until drying.
41. Dry down the combined eluates using a vacuum centrifuge
E. LC-MS sample preparation & LC settings
42. Dissolve dried samples (step 41) in 225 µl/tube of LC Solution
by pipetting up and down.
43. Vortex the tubes thoroughly or shake them on thermomixer for
10 min at room temperature.
44. Centrifuge the dissolved samples at 4°C and 20`000 x
g
for
20 min.
45. Transfer 6 µl of sample supernatants to LC-MS vials. Store
remaining samples at -20°C.
46. Prepare Reference Peptide Mix by adding to the glass vial
10 µl of Dissolution Buffer; vortex briefly.
Note 10
You may increase the
centrifugation force up
to 400 x g in case of low
flow rates through the
MicroSpin columns. The
force can be increased
to max 1’000 x g.
Note 11
Dried samples can be
stored at -20°C until
usage.
