Biology Reference
In-Depth Information
5. 13 and 14 gauge, Kel-F Hub NDL, 2 in., point style 3 (Part
Numbers 7750-04 and 7750-05, Hamilton) needles to punch
out small disks for preparing 1 ml pipet tip used in StageTip
columns.
6. Plunger Assemblies (Part Numbers 1122-01 and 1162-01,
Hamilton): 1122-01 and 1162-01 are for 16 gauge and 13/14
gauge needles, respectively.
7. Low binding 200
l and 1 ml pipet tips.
8. Lids of micro-centrifuge tubes.
9. Lids of 15 ml conical tubes.
10. Sharp-ended tweezers to drill a hole in the lids of micro-
centrifuge tubes.
11. Soldering iron or drill to drill a hole in 15 ml conical tube lids.
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1. C18 1 ml StageTip: 1 ml pipet tip packed with four C18 disks
with the 14 gauge needle and two C18 disks with the 13 gauge
needle. Can be kept at room temperature.
2. 15 ml Conical tube.
3. The spin adaptor for 15 ml conical tube.
4. 2 % trifl uoroacetic acid (TFA): 2 % TFA in water.
2.3 Desalt the
Digests with C18 1 ml
StageTip Column
5. Solution A: 80 % acetonitrile (ACN) and 0.1 % TFA in water.
6. Solution B: 5 % ACN and 0.1 % TFA in water.
7. SpeedVac.
Some types of StageTips are commercially available.
2.4 HAMMOC
StageTip Column
Preparation
1. C8 200
l pipet tip packed with a single C8
disk made with the 16 gauge needle. Can be kept at room
temperature.
2. Titania (TiO
2
) beads: Titansphere Bulk Media, 10
μ
l StageTip: 200
μ
m (Part
number 5020-75010, GL science). Store under dry conditions
to keep specifi city against phosphopeptides (
see
Note 2
). The
specifi city varies to some extent from one production lot to
another.
3. Methanol.
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2.5 Phosphopeptide
Enrichment
by HAMMOC
1. Solution C: 300 mg/ml lactic acid in solution A.
2. HAMMOC StageTip: The C8 200
l StageTip with 3 mg
TiO
2
beads. Prepare just before use (
see
Note 2
).
3. Micro-centrifuge tube.
4. The spin adaptor for micro-centrifuge tube.
5. Solution A: 80 % ACN and 0.1 % TFA in water.
μ
