Biology Reference
In-Depth Information
1. Add 400
L MeOH to each well to activate and wash the
SPEC plate and repeat once (use one well for each sample).
2. Equilibrate four times with 400
μ
L dH 2 O.
3. Meanwhile spin down trypsin beads by centrifugation at
14,000 × g for 10 min at RT.
4. Load 500
μ
L peptides-containing supernatant to the center of
the well and allow the column to absorb the peptides by incu-
bating for 1 min.
5. Repeat step 4 until each sample has been completely loaded
onto a single well.
6. Wash four times with 400
μ
L dH 2 O.
7. Change collection plate and use an Eppendorf Protein LoBind
96-deepwell plate to collect peptides.
8. Elute peptides with 200
μ
μ
L MeOH by incubating for 5 min
and repeat once.
9. Transfer the peptide solution (eluate) to a 1.5 mL Eppendorf
Protein LoBind tube using a glass Pasteur pipette.
10. Dry peptides completely in a speedvac.
3.4 TiO 2 -Based
MOAC Enrichment of
Phosphopeptides
1. Dissolve peptides in 100
μ
L B1 and centrifuge for 10 min at
12,000 × g .
2. For each sample containing 500
g peptides weigh 12.5 mg
TiO 2 into a spin column with a polyethylene fi lter of 10
μ
μ
m
pore size, screw cap and press-in bottom plug.
3. Equilibrate TiO 2 by adding 250
μ
L B1 to each column and
incubating for 5 min.
4. Spin column at 700 × g for 2 min.
5. Add peptide mixture of step 1 to the column and allow phospho-
peptides to bind the TiO 2 chromatography media for 15 min by
closing the column and incubating it head-over-head.
6. Open the column and place it in a clean tube. Spin at 700 × g
for 2 min, the fl ow-through fraction containing non-
phosphorylated peptides can be desalted and stored at −20 °C
for further analysis.
7. The column is then washed six times in total using 250
L
buffer for each washing step: 2× B1, 2× B2 and then 2× B3.
8. Phosphopeptides are eluted with 100
μ
L B4 by incubating the
closed column for 5 min head-over-head.
9. Collect the eluted phosphopeptides in a clean 1.5 mL
Eppendorf Protein LoBind tube by centrifugation at 700 × g
for 2 min.
10. Dry peptides completely in a speedvac.
μ
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