Supplemental Protocol: Yeast Lysis with PIXUL™ Multi-Sample Sonicator
Description: PIXUL Multi-Sample Sonicator makes yeast lysis easy.
IMPORTANT: PIXUL sonication requires using PIXUL 96-well round bottom plates (cat. no. 53139).

1.

Add 500 μl 5% SDS 50 mM TEAB to yeast pellets derived from 2 ml overnight cultures. Measure pH which shouldbe 7-8. Buffer volume can be less for smaller pellets.

2.

Load 100 μl sample in each well of 96-well microplate, seal, and sonicate in PIXUL for 15-30 minutes (min).

3.

PIXUL parameters: ulse Pulse N=50, PRF=1 kHz, Process Time = 30 or 60 minutes, Burst Rate=20 Hz.

4.

Quick-spin 96-well microplate. Transfer sonicated lysates into centrifuge tubes. Go to step 9.

5.

Or, centrifuge 96-well microplate at maximum speed for 15 minutes, extract only supernatant. Go to step 10.

6.

To compare PIXUL in parallel with probe sonicator, afterwith lysing matrix Y.

7.

Lyse in Qiagen tissue lyser for 5 min, 30 Hz. Keep aside 10 μl aliquot for agarose gel.

8.

Perform bead beating followed by probe sonication: 3 cycles x 10 seconds, 30s on ice in between cycles.

9.

Centrifuge for 15 min at maximum speed i.e. 13.2k rpm.

step 1, transfer lysate of 3 tubes to centrifuge tube

10. Keep aside 10 μl aliquot for agarose gel.
11.

Perform BCA assay.
a. Dilute sample 16x (2.5 μl sample + 37.5 μl 5% SDS 50 mM TEAB)
b. Use 10 μl of diluted sample per replicate in 200 μl WR (50:1, A:B)

12. Quantify protein extraction in μg, isolate 100 μg protein extract.
13. Adjust volume to 50 μl and 5% SDS 50 mM TEAB f.c.
14. Add 1.5 μl 0.5 M DTT, 30 min @55°C.
15. Add 3 μl 0.5 M IAA, 15 min @RT in dark.
16. Add 5 μl 12% phosphoric acid, check pH<1. If pH>1, add more phosphoric acid.
17.

Add 350 μl 90% MeOH 100 mM TEAB pH 7.55.

18. Pipet samples on S-trap plate, centrifuge for 2 min at 1500xg.
19. Wash 3 times with 200 μl 90% MeOH 100 mM TEAB pH 7.55, centrifuge for 2 min at 1500xg.
20. Add 125 μl 50 mM TEAB containing 1 μg Trypsin (1:100).
21. Overnight incubation in 37°C, cover loosely with the cover supplied with S-trap plates.
22. Add 80 μl 50 mM TEAB, centrifuge for 2 min at 1500xg.
23. Add 80 μl 0.2% formic acid, centrifuge for 2 min at 1500xg.
24. Add 80 μl 50% ACN, 0.2% formic acid, centrifuge for 2 min at 1500xg.
25. Transfer to MS vials, speedvac and store at -20°C.
Protocol courtesy of Vlaams Instituut voor Biotechnologie (VIB), Belgium; VIB Proteomics Core

activemotif.com

Technical Support

If you need assistance at any time, please call or send an e-mail to Active Motif Technical Service at one of the locations listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008, USA

Active Motif Europe
Waterloo Atrium
Dréve Richelle 167 – boîte 4BE-1410 Waterloo, Belgium

Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Active Motif China
290 Wankang Road
Suite 601
Minhang District
Shanghai, 201112, China

Toll Free: 877 222 9543
Direct: 760 431 1263
Fax: 760 431 1351
E-mail: tech_service@activemotif.com

Germany Free Phone: 0800 181 99 10
France Free Phone: 0800 90 99 79
UK Free Phone: 0800 169 31 47
Fax: +32 (0)2 653 0050
E-mail: eurotech@activemotif.com

Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
E-mail: japantech@activemotif.com

Direct: (86)-21-20926090
Cell Phone: 18521362870
E-mail: techchina@activemotif.com

Visit Active Motif online at http://www.activemotif.com

activemotif.com