2.3 Ultracentrifugation and Sample
Concentration
1. Ultracentrifuge.
2. Thickwall polycarbonate tube (8Â 34 mm, 343776).
3. Tabletop centrifuge.
4. Pierce™ BCA assay kit (Thermo Fisher Scientific).
5. Amicon
® molecular cutoff filter (30 kDa MWCO) with cellulose membrane.
6. Protein LoBind Tubes (Eppendorf
® ).
7. Snap/Crimp/Vial PP 12 Â 32 mm, with 11 mm Snap-Cap
w. Silicone/PTFE-Septum (Septa).
2.4 HPLC Standard
Operation Procedure
1. Filtered SEC running buffer, HN-buffer, pH 7.5: 50 mM
HEPES, 150 mM NaCl, store till usage at 4
C (see Note 2).
2. High MW protein mixture standards (AL0-3042 Column Performance Check Standard, Phenomenex), store till usage at
4
C.
3. HPLC system with temperature control at 4
C. The system is
set up with degasser, sample injection loop, UV detector, and
analytical scale fraction collector. Specifications for all HPLC
modules used are reported in Table 1. The experiment can be
performed with similar or equivalents devices.
4. The separation can be performed with different column materials. The choice of column and pre-column can be made
according to column capacity or linear molecular weight separation range of interest. To increase column life, we recommend always to use a pre-column, according to the column
manufacturing information for the choice of the best suitable
pre-column. For the projects in our research group, we
employed various different column materials from different
manufacturers (see Table 2).
5. 96-Well Filter Plates (AcroPrep Advance Filter Plates for Ultrafiltration 350 μL, Omega membrane, 10K MWCO,
8034, PALL).
6. HPLC gradient grade water (Fisher Chemical, W/0106/17),
referred to as H 2 O in the protocol.
2.5 High Throughput
Filter-Aided Sample
Preparation Protocol
of SEC-Fraction
for MS-Analysis (FASP
Protocol)
1. Proteolysis buffer: prepare fresh the following buffers:
(a) Buffer TUA: 8 M Urea, 20 mM ammonium bicarbonate,
5 mM TCEP.
(b) Buffer IA: 50 mM iodoacetamide, 20 mM ammonium
bicarbonate.
(c) Buffer ABC: 20 mM ammonium bicarbonate.
2. Sequencing grade trypsin (Promega V511).
3. Lysyl Endopeptidase
® , Mass Spectrometry grade (Wako).
System-Wide Profiling of Protein Complexes Via Size Exclusion. . .
273
Concentration
1. Ultracentrifuge.
2. Thickwall polycarbonate tube (8Â 34 mm, 343776).
3. Tabletop centrifuge.
4. Pierce™ BCA assay kit (Thermo Fisher Scientific).
5. Amicon
® molecular cutoff filter (30 kDa MWCO) with cellulose membrane.
6. Protein LoBind Tubes (Eppendorf
® ).
7. Snap/Crimp/Vial PP 12 Â 32 mm, with 11 mm Snap-Cap
w. Silicone/PTFE-Septum (Septa).
2.4 HPLC Standard
Operation Procedure
1. Filtered SEC running buffer, HN-buffer, pH 7.5: 50 mM
HEPES, 150 mM NaCl, store till usage at 4
C (see Note 2).
2. High MW protein mixture standards (AL0-3042 Column Performance Check Standard, Phenomenex), store till usage at
4
C.
3. HPLC system with temperature control at 4
C. The system is
set up with degasser, sample injection loop, UV detector, and
analytical scale fraction collector. Specifications for all HPLC
modules used are reported in Table 1. The experiment can be
performed with similar or equivalents devices.
4. The separation can be performed with different column materials. The choice of column and pre-column can be made
according to column capacity or linear molecular weight separation range of interest. To increase column life, we recommend always to use a pre-column, according to the column
manufacturing information for the choice of the best suitable
pre-column. For the projects in our research group, we
employed various different column materials from different
manufacturers (see Table 2).
5. 96-Well Filter Plates (AcroPrep Advance Filter Plates for Ultrafiltration 350 μL, Omega membrane, 10K MWCO,
8034, PALL).
6. HPLC gradient grade water (Fisher Chemical, W/0106/17),
referred to as H 2 O in the protocol.
2.5 High Throughput
Filter-Aided Sample
Preparation Protocol
of SEC-Fraction
for MS-Analysis (FASP
Protocol)
1. Proteolysis buffer: prepare fresh the following buffers:
(a) Buffer TUA: 8 M Urea, 20 mM ammonium bicarbonate,
5 mM TCEP.
(b) Buffer IA: 50 mM iodoacetamide, 20 mM ammonium
bicarbonate.
(c) Buffer ABC: 20 mM ammonium bicarbonate.
2. Sequencing grade trypsin (Promega V511).
3. Lysyl Endopeptidase
® , Mass Spectrometry grade (Wako).
System-Wide Profiling of Protein Complexes Via Size Exclusion. . .
273