Cells were lysed in NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl (pH 8.0), 0.5% Nonidet P-40) containing protease inhibitors (Roche). For pulldown of SFB-tagged proteins or MYC-tagged proteins, cell extracts were incubated with streptavidin–Sepharose beads (Amersham Biosciences) or Anti-Myc magnetic beads (Thermo Fisher, 88842) at 4 °C for 2 h. The beads was washed with NETN buffer and the bound proteins were eluted by boiling in 1× Laemmli buffer.
Anti myc magnetic beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
Anti-myc magnetic beads are a laboratory tool designed for the purification and detection of proteins tagged with the c-myc epitope. These beads contain immobilized anti-c-myc antibodies that can selectively bind to and capture c-myc-tagged proteins from complex samples, enabling their isolation and further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Protein inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Anti flag m2 magnetic bead, by Merck Group (2 mentions)
Polyjet, by SignaGen (2 mentions)
Propidium iodide, by Thermo Fisher Scientific (2 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (2 mentions)
Cacl2, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions)
Biotin azide, by Cayman Chemical (2 mentions)
Dii c12, by Thermo Fisher Scientific (2 mentions)
Anti myc alexafluora 647, by Cell Signaling Technology (2 mentions)
Tris 2 carboxyethyl phosphine tcep, by Research Products International (2 mentions)
Tris 1 benzyl 1h 1 2 3 triazol 4 yl methyl amine tbta, by Thermo Fisher Scientific (2 mentions)
Anti myc, by Cell Signaling Technology (2 mentions)
4 2 hydroxyethyl 1 piperazineethanesulfonic acid hepes, by Research Products International (2 mentions)
Cucl2, by Thermo Fisher Scientific (2 mentions)
Dithiothreitol (dtt), by Research Products International (2 mentions)
Hela cell line, by American Type Culture Collection (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Streptavidin sepharose beads, by Cytiva (1 mentions)
17 protocols using anti myc magnetic beads
1
Pulldown of tagged proteins
2
Protein Interaction Profiling in HEK293T Cells
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The indicated plasmids were transfected into HEK293T cells. The cells were collected and lysed at 4 °C for 30 min in IP lysis buffer (1% NP-40, 0.025 M Tris-HCl, 0.15 M NaCl, 1 mM EDTA, 5% glycerol) supplemented with phosphatase inhibitor/PMSF, followed by centrifugation for 10 min at 12,000 × rpm under 4 °C. We retained the supernatants, one third of which was used for input analysis and the other two thirds were incubated with Anti-Myc Magnetic beads (Pierce #88842, Thermo Fisher Scientific, Waltham, MA, USA), or IgG control, overnight at 4 °C for IP analysis. IP lysis buffer was then used to wash the precipitates three times, followed by boiling the samples in 2 × loading buffer. Western blotting was then used to analyze the precipitates using the indicated primary antibodies, followed by incubation with HRP-conjugated anti-rabbit IgG (conformation specific) (#5127, Cell Signaling Technology) or anti-mouse IgG (Light Chain Specific) (#58,802, Cell Signaling Technology) secondary antibodies. The immune-reactive proteins were visualized using the ECL reagent.
3
Immunoprecipitation and Protein Analysis
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Cells were washed on the plate with PBS and lysed in Pierce immunoprecipitation lysis buffer (Thermo Fisher Scientific, PI87787) supplemented with Pierce Halt protease and phosphatase inhibitor cocktail (no. 78443). Immunoprecipitation was performed by incubating protein lysates with antibody-coupled magnetic beads (anti-HA magnetic beads [Cell Signaling Technology, 11846S] and anti-myc magnetic beads [Thermo Fisher Scientific, PI88842]) overnight at 4°C with rotation followed by three washes with lysis buffer. Samples were either brought for mass spectrometry analysis or eluted with SDS-PAGE gel loading buffer (Thermo Fisher Scientific, 50194470) for Western blot analysis; otherwise, cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitor cocktail for direct Western blot analysis.
4
RNA-Protein Binding Assay
5
Recombinant Protein Expression and Purification
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Recombinant protein expression in mammalian cells and protein purification were conducted as previously described (27 (link)). Briefly, 293T cells were transfected with 3xFlag-hDNA2 or myc-hTRAF6 using the Polyjet (SignaGen) transfection reagent. Twenty-four hours after transfection, the cells were collected and lysed by brief sonication in the immunoprecipitation (IP) buffer H150 (50 mM HEPES-KOH [pH7.4], 150 mM NaCl, 0.1% NP40 and 10% glycerol) with protein inhibitor cocktail (Thermo Fisher). After centrifugation (20,000 × g, 15 min, 4°C), the supernatant was incubated with anti-Flag M2 magnetic beads (Sigma) or anti-myc magnetic beads (Thermo Fisher) overnight. The beads were washed with H500 buffer (50 mM HEPES-KOH [pH7.4], 500 mM NaCl, 0.1% NP40 and 10% glycerol) and H150 buffer (50 mM HEPES-KOH [pH7.4], 150 mM NaCl, 0.1% NP40 and 10% glycerol) once, then eluted with 250 μg/ml 3xFlag peptide or myc peptide (Apex) for 6 h.
6
Immunoprecipitation of Flavivirus Capsids
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Mock and stably transduced A459 cells expressing AcGFP alone or AcGFP and myc-tagged ZIKV capsid (1 × 106) were lysed in 400 mL IP buffer (50 mM Tris-HCL (pH 7.6), 20 mM NaCl, 1 mM MgCL2, 1% Triton X-100, 1× cOmplete™ mini EDTA-free protease inhibitor cocktail (Roche, Indianapolis, USA) and 1× PhosSTOP (Roche) for 15 min at 4 °C with rotation, and then clarified by centrifugation at 14,000 rpm for 10 min. The cleared lysates were incubated with anti-myc magnetic beads (Thermo-Scientific) overnight with rotation at 4 °C. The beads were washed twice with 400 mL IP buffer before bound proteins were eluted by heating at 95 °C for 5 min in 40 μL of 2× Laemmli sample buffer containing 5% β-mercaptoethanol, followed by SDS-PAGE and immunoblotting. Where indicated, A459 cells transiently expressing myc-tagged ZIKV, YFV, DENV, WNV, JEV or SLEV capsids were processed in the same manner at forty-hours post-transfection.
7
Kinase Assay of TGF-β1 and PRKACA
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The in vitro kinase assay was performed as described previously51 (link). Briefly, Flag-TGF-β1WT or Flag-TGF-β1T282A was transiently overexpressed with Myc-PRKACA in HEK293T cells and purified using anti-Flag magnetic beads or anti-Myc magnetic beads (88842, Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s protocol. The precipitated Flag-TGF-β1, Flag-TGF-β1T282A, and Myc-PRKACA proteins were resuspended in 40 μl of 1 × kinase buffer (9802) supplemented with 200 μM ATP (9804) (Cell Signaling). The reaction was carried out for 30 min at 30 °C and was terminated by the addition of 20 μl 3 × SDS sample buffer. Each sample was then boiled for 10 min at 100 °C and subjected to SDS–PAGE and IB analysis using the indicated antibodies.
8
Interactome Analysis of hDNA2 and hTRAF6
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293T cells were transfected with 3xFlag-hDNA2 or myc-hTRAF6 alone or in combination using the Polyjet (SignaGen) transfection reagent. Twenty-four hours after transfection, the cells were collected and lysed by brief sonication in the IP buffer H150 with protein inhibitor cocktail (Thermo Fisher). After centrifugation (20,000 × g, 15 min, 4°C), the supernatant was incubated with anti-Flag M2 magnetic beads (Sigma) or anti-myc magnetic beads (Thermo Fisher) overnight. The beads were washed three times with H150 buffer, and then eluted with 250 μg/ml 3xFlag peptide or myc peptide (Apex) for 6 h. The 3xFlag-hDNA2 or myc-hTRAF6 was subjected for analysis by western blot analysis or mass spectrometry.
To immunoprecipitate endogenous hDNA2, HeLa cell lysate was incubated with an anti-hDNA2 antibody and Protein A/G dynabeads in the IP buffer overnight. The beads were washed three times with H500 buffer and subjected to boiling in 1× sodium dodecyl sulphate-polyacrylamide gelelectrophoresis (SDS-PAGE) loading buffer directly. hDNA2 levels were analyzed by western blot analysis.
To immunoprecipitate endogenous hDNA2, HeLa cell lysate was incubated with an anti-hDNA2 antibody and Protein A/G dynabeads in the IP buffer overnight. The beads were washed three times with H500 buffer and subjected to boiling in 1× sodium dodecyl sulphate-polyacrylamide gelelectrophoresis (SDS-PAGE) loading buffer directly. hDNA2 levels were analyzed by western blot analysis.
9
Co-immunoprecipitation of SmCSP4-HA and TaWRKY76-MYC
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SmCSP4‐HA and TaWRKY76‐MYC were co‐expressed in N. benthamiana leaves, and total protein was extracted from these leaves using the RIPA Lysis Buffer (Beyotime, China). Protein extracts were centrifuged at 15000g at 4 °C for 30 min and the supernatants were incubated with anti‐HA magnetic beads or anti‐MYC magnetic beads (Thermo Fisher, USA) for 3 h at 4 °C. The Co‐IP assay was performed using the Pierce Magnetic HA‐Tag IP/Co‐IP Kit and Pierce Magnetic MYC‐Tag IP/Co‐IP Kit (Thermo Fisher, USA). The immunoprecipitated proteins were detected via Western blotting using anti‐MYC (1 : 1000) antibody or anti‐HA antibody (1 : 1000) (antibodies‐online Inc., Germany). The luminescent signal was visualized using BIO‐RAD ChemiDoc™XRS+. N. benthamiana leaves co‐expressed with TaWRKY76‐MYC and HA were set as controls, and IP with IgG antibody served as negative control.
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SmCSP4‐HA and TaWRKY76‐MYC were co‐expressed in N. benthamiana leaves, and total protein was extracted from these leaves using the RIPA Lysis Buffer (Beyotime, China). Protein extracts were centrifuged at 15000
10
STING-HA Immunoprecipitation and Western Blot
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The STING-HA expression plasmids were kindly provided by Dr. Russell E. Vance1 (University of California, Berkeley, California) and have been previously described [69 (link)]. CD40-Myc was cloned into pCMV6-entry vector as described below. For immunoprecipitation, whole-cell extracts were prepared after transfection or stimulation with appropriate ligands, followed by incubation with anti-HA or anti-Myc magnetic beads (Thermo Fisher Scientific, Rockville, MD). Co-IP was performed according to the manufacturer’s instructions and subsequently analyzed on Western blot. Cell lysates or immunoprecipitated complexes were separated on SDS-PAGE gels and transferred to PVDF membranes (Thermo Fisher Scientific). Membranes were incubated with primary antibodies followed by HRP-conjugated secondary antibodies. Bound HRP-labeled antibodies were visualized using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific).
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