Biology Reference
In-Depth Information
Put solution into
SPE C-TIP
Solution passes
through column by
centrifugation
Fig.
3
Peptide purifi cation assembly. A C-TIP is inserted in a hole drilled with a
soldering iron in a 1.5 mL microtube cap. Solution A, solution B, and the sample
solution are put into the C-TIP, in that order, and then the assembly is centrifuged.
The mixed solution passes through a C18 column and peptides are absorbed on,
or eluted from, the C-TIP
3.5 Peptide
Purifi cation
All of these procedures must be performed at a clean bench when-
ever possible and at room temperature unless otherwise specifi ed.
1. Insert a SPE C-TIP into the 3 mm hole in the microtube top
(Fig.
3
).
2. Add 30
L of solution A to the upper side of the SPE C-TIP
for preconditioning. Centrifuge at 1,000 ×
g
for 30 s to get
solution A through the tip column.
3. Add 30
μ
L of solution B from upper side of SPE C-TIP for
preconditioning. Centrifuge at 1,000 ×
g
for 30 s to get solu-
tion B through the tip column.
4. After confi rming that the column is moist, add the entire tryp-
sin-digested peptide sample to the upper side of the SPE
C-TIP for column absorption. Centrifuge at 1,000 ×
g
for 30 s
to get the sample solution through the tip column.
5. Add 30
μ
L of solution B from upper side of SPE C-TIP for
cleaning. Centrifuge at 1,000 ×
g
for 30 s to get solution B
through the tip column.
6. Put a vial insert for each LC-MS/MS sampler into another
holed microtube. Transfer the SPE C-TIP into the
microtube.
7. Add 30
μ
L of solution A to the upper side of the SPE C-TIP
for elution. Centrifuge at 1,000 ×
g
for 30 s to get solution A
through the tip column. Discard the SPE C-TIP.
8. Dry out the eluted samples using a centrifugal concentrator
for 15 min. Add 15
μ
L of 0.1 % (v/v) TFA. Put the vial insert
into the vial and close the lid. Store at −30 °C (
see
Note 23
).
μ
