Protein a sepharose 4 fast flow beads

RMS cells were lysed in ice-cold lysis buffer (200 mM Tris, pH7.4; 120 mM NaCl; 0.2% sodium deoxycholate; 0.1% SDS; 1 mM EGTA, 1% Triton-X 100) supplemented with 1× protease inhibitor (Sigma; Cat# P8340). Debris was removed by centrifugation at 6000 rpm for 5 min at 4 °C and the lysates obtained were quantified using BCA Protein Assay kit (Pierce, Thermo Fisher Scientific; Cat# 23225) as per manufacturer’s protocol. Approximately 500 µg protein was incubated with Protein A Sepharose 4 Fast flow beads (GE Healthcare Life Sciences, Chicago, IL, USA; Cat# 17-5280-01) for 1-h at 4 °C on a tube mixer, followed by centrifugation to remove the beads and the proteins that were bound non-specifically. Subsequently, the pre-cleared lysates were used to immunoprecipitate MET-conjugated proteins by incubation with MET antibody overnight at 4 °C on a tube mixer. IgG raised in rabbit was used as a control antibody. The antibody-bound proteins were then incubated with Protein A Sepharose 4 Fast flow beads for 6-h at 4 °C on a tube mixer to capture the immunocomplexes. Immunoprecipitates were washed thrice using ice-cold lysis buffer and eluted by boiling in 2× Laemmli sample buffer at 95 °C for 3 min. The immunoprecipitates were subjected to western analysis as described below. Antibodies and the concentrations used for immunoprecipitation are listed in Supplementary Table 2.

Protein A Sepharose 4 Fast Flow beads (GE Healthcare) were washed thrice at 1200 rpm with 1X PBS. 10 µl rabbit anti-GUS antibody (Sigma Aldrich, catalog number G5420) was added in 200 µl washed protein A beads in 1X PBS and allowed to bound at 4 °C for 4 h under shaking. The antibody-protein A bead complex was washed thrice with 1X PBS. The total protein from the tobacco transplastomic plant leaf was added to the antibody-protein A complex and incubated in a cold room overnight under shaking. Three washes were performed with 1X PBS at 1200 rpm for 5 min. The antigen (GUS protein)-antibody-protein A bead complex was resuspended in 15 µl 4X SDS PAGE sample buffer by boiling. After immunoprecipitation, the samples were separated on 10% SDS–PAGE mini gels as described in the instruction manual for the Mini-Protean III Electrophoresis Cell (BioRad).

Cells were lysed in lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.0,1 mM EDTA, 0.5% Triton-100 (for HeLa cell); 150 mM NaCl，50 mM Tris-HCl pH 8.0, 10 mM NaF, 1 mM Na₃VO_4,1% NP-40, 10% glycerol, 1.5 mM EDTA pH 8.0 (for S2 cell)), supplemented with protease inhibitors PMSF, aprotinin, pepstatin, leupeptin, and phosphatase inhibitor PhosSTOP (sigma, 4906837001). Samples were centrifuged at 16000 g for 10 min and supernatant were collected and incubated with HA beads (Sigma, E6779) or Flag beads (Sigma, A2220) or V5 antibody followed with Protein A Sepharose 4 Fast Flow beads (GE Healthcare, 17-5280-01) for 2–4 hr at 4°C. Spin down the beats at 500 g for 30 s. Then washed with lysis buffer for three times and add SDS running buffer to the beads and proceed to western blot analysis. For western blotting analysis, proteins were separated by SDS–PAGE, and transferred onto a PVDF membrane. The membrane was then blocked with 5% non-fat milk (Sangon Biotech, A600669) in TBST buffer and incubated with primary antibodies in TBST with 5% non-fat milk overnight at 4°C. The membranes were then washed in TBST and incubated with HRP labeled secondary antibodies (1:5000 in TBST with 5% non-fat milk) for 1 hr at RT. The membranes were then washed in TBST and developed with ECL reagents (Cyanagen Srl, XLS3-0020) and exposed. Quantification of protein bands was done with Image J software.

HeLa P4 cells were washed twice with cold PBS containing 0.1 mM CaCl₂ and 1 mM MgCl₂ and incubated with dithiobis(succinimidyl propionate); (DSP; Thermo Scientific) at a final concentration of 1 mM in DMSO for 2 h on ice. For control reactions, DMSO alone was used. DSP was quenched by the addition of 20 mM Tris-HCl, pH 7.4, for 15 min. The cells were then washed twice with cold PBS and lysed with 1 mL of lysis buffer (0.5% sodium deoxycholate, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.25% SDS, and 0.5% Triton X-100 with Complete protease inhibitor mixture (Roche Applied Science)) for 30 min on ice. To reduce viscosity, the lysate was passed through a 27-gauge×3/4-inch needle and then centrifuged at 15,000 g for 20 min at 4 °C. For immunoprecipitation of endogenous protein complexes, 3 µg of mouse anti-emerin, or IgG as a control were immobilized on 50 µL of Protein A-Sepharose 4 Fast Flow beads (GE Healthcare) for 3 h and incubated with lysates from 24 × 10⁶ cells that had or had not been subjected to cross-linking as described above. The beads were then washed four times with washing buffer (10 mM HEPES, 150 mM NaCl, 1 mM EGTA, 0.1 mM MgCl₂, 0.1% Triton X-100, and Complete protease inhibitor mixture), and proteins were eluted with sample buffer containing 50 mM DTT.

Protein A Sepharose 4 Fast Flow Beads (GE Healthcare #17-5280-01) were washed with 1mL of ice-cold 1x PBS and then blocked with 0.1% BSA in PBS for 1 hour while rotating at 4°C. Beads were washed twice with 1 mL ice-cold PBS and once with Isotonic Wash Buffer (20 mM Tris-HCl pH 7.5, 150mM NaCl, 0.1% NP-40). Beads were conjugated with the indicated antibodies or immunoglobulin (IgG) at a dilution of 1:125 in 1mL of Isotonic Wash Buffer for 3 hours at 4°C while rotating. Beads were then washed twice with 1mL of ice-cold Isotonic Wash Buffer.

After identification by MALDI-TOF-MS, validation was performed by immunodepletion using specific antibodies against the protein identified. 500 µL of RIPA buffer (Triton X-100 1%, NaCl 150 mmol/L, EDTA 1 mmol/L, EGTA 1 mmol/L, sodium vanadate 0.1 mmol/L, and NP40 0.5% in Tris-HCl 10 mmol/L) was added by 1 µL of native plasma sample and 5 or 10 µg of antibodies, as previously described [22] (link). The antibodies used were for haptoglobin (SantaCruz sc-134466), hemoglobin (SantaCruz sc-31116), apolipoprotein C-III (Tebu-bio, 036sc-50378), transthyretin (Abcam, ab905), apolipoprotein A-IV (SantaCruz sc-19036) and fibrinogen (sc-335581). After overnight incubation at 4°C, 50 µL of protein A Sepharose 4 Fast Flow beads (GE Healthcare) were added and the preparation was rotated end-to-end 4 h at 4°C. Supernatant was next obtained after centrifugation, and was precipitated in cold acetone overnight. Supernatant was removed after centrifugation 20 min at 12,000 g and pellet was dried 5 min at room temperature and added by 5 µL of SELDI binding buffer (depending on the array). Immunodepleted and undepleted plasma were compared by SELDI-TOF-MS on ProteinChip CM10 or H50 arrays and disappearance of the peak of interest was visualized, compare to untreated plasma.