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PAC Workflow

Protein capture

Add beads

MagStart-Amine (70% ACN)

25 min

Add protein extract

5 to 1000 μg in 5% SDS 50 mM Tris pH 8 (1:5 protein to bead ratio)

Add ACN

Final concentration of 70%

Mix

1 min

Pause

10 min

Repeat mix & pause cycle

1×cycle

collect

Bead-Protein aggregate captured

Sample washing

transfer bead-protein aggregate to wash plate

1 ml 95% CAN (3 plates) or 70% EtOH (2 plates)

12.5 min

Mix gently with bead-protein aggregate on magnet

2.5 min

Cycle

5×wash cycle

Digest

Transfer aggregate to digest plate & release

250 μl 50 mM AmmBic and digest enzymes

60~720 min

Incubation (37~47℃，1~12h) with intermittent mixing

Loop: mix 15sec, pause 2 min 15 sec for duration of digest

Quench digest and mix

1% TFA

Collect beads on magnetic

Bead storage

Transfer beads to storage

500 μl 70% ACN

1 min

Release beads from magnet

Phosphopeptide Enrichment Workflow

Bead equilibration

Aliquote beads

MagStart-IMAC-Ti/Zr, 40 μl  (800μg)

5 min

Collect beads to transfer beads to equilibration plate

500 μl 80% ACN, 5% TFA, 0.1~1 M Glycolic acid

Phosphopeptide binding

Transfer beads to desalted digest

200 μg in 80% ACN, 5% TFA, 0.1~1 M Glycolic acid

20 min

Mix gently to ensure peptide sample interaction

KF medium speed

Sample washing

Transfer beads with bound phosphopeptides to wash plate 1

Wash 3: 500 μl 80% ACN, 5% TFA, 0.1~1 M Glycolic acid

6min

Mix

2 min

Transfer beads with bound phosphopeptides to wash plate 2

Wash 3: 500 μl 80% ACN, 1% TFA

Mix

2 min

Transfer beads with bound phosphopeptides to wash plate 3

Wash 3: 500 μl 10% ACN, 0.1% TFA

Mix

2 min

Elution

Capture beads with phosphopeptides and transfer to elution plate

200 μl 1% NH₄OH

10 min

Bead storage

Transfer beads to storage

1 min

Release beads in storage buffer

20% EtOH