Immunoprecipitation from yeast cells was carried out on extracts prepared by glass bead lysis. Immunoprecipitation was performed from human cell extracts generated as described below or from combining the Chromatin I and II fractions (described in cell fractionation) to form the Chromatin fraction. Typically, 1 mL extract at 1 mg/mL was incubated with 50 μL beads alone for 1 h at 4°C to pre-clear non-specific binding. GIGYF2 (A303-732) (Bethyl Laboratories Inc. Texas), proteasome (ab109530) (Abcam), HA (12CA5) or myc (9E10) antibodies were pre-incubated with protein A or G beads for 1 h at 4°C before incubation with the pre-cleared extract. M2 anti-flag agarose (Sigma-Aldrich 30410) was used for FLAG tagged purifications. 50 μL of antibody bound beads were rotated with the extracts for 3 h at 4°C. Unbound material was saved and the beads were either washed two times in their immunoprecipitation buffer, followed by once in high salt (500 mM) and then washed back into the applicable immunoprecipitation buffer. Beads were eluted by the addition of 2.5x SDS-loading buffer.
Anti flag m2 agarose
Manufactured by Merck Group
Sourced in United States, Germany
The Anti-FLAG M2 agarose is a laboratory reagent used for the purification and detection of proteins that have been tagged with the FLAG peptide sequence. It is a bead-based affinity matrix that binds to the FLAG tag with high specificity, allowing for the efficient capture and recovery of the targeted protein.
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Lab products found in correlation
Flag peptide, by Merck Group (25 mentions)
Anti ha agarose, by Merck Group (11 mentions)
3xflag peptide, by Merck Group (10 mentions)
Protease inhibitor cocktail, by Roche (7 mentions)
Ni nta agarose, by Qiagen (6 mentions)
Anti flag, by Merck Group (6 mentions)
Complete protease inhibitor cocktail, by Roche (5 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Mouse igg agarose, by Merck Group (3 mentions)
Freestyle 293 expression system, by Thermo Fisher Scientific (3 mentions)
Proteinase and phosphatase inhibitor, by Roche (3 mentions)
Complete protease inhibitor, by Roche (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (3 mentions)
Expifectamine, by Thermo Fisher Scientific (2 mentions)
Expi293 cell, by Thermo Fisher Scientific (2 mentions)
Gfp trap agarose, by Proteintech (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
219 protocols using anti flag m2 agarose
1
Immunoprecipitation from Yeast and Human Cells
2
Purification of PI4KIIIα protein complexes
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Expi293 cells (Thermo Fisher) were maintained and transfected with ExpiFectamine (Thermo Fisher), according to the manufacturer’s instructions, with the following combinations of plasmids: (1) 3xFLAG-PI4KIIIα (wild-type); (2) 3xFLAG-PI4KIIIα (D1957A kinase-dead mutant); (3) 3xFLAG-PI4KIIIα (WT) and TTC7B-HA; or (4) 3xFLAG-PI4KIIIα (WT), TTC7B-HA, and GFP-FAM126A(1-289). Cells were harvested, resuspended in lysis buffer (250 mM NaCl, 20 mM Tris pH 8.0, 10% glycerol, 1 mM TCEP, supplemented with protease inhibitors (cOmplete, EDTA-free (Roche))), sonicated briefly, and centrifuged for 10 min at 16,000 × g. The supernatant was immunoprecipitated in lysis buffer by addition of M2 anti-FLAG agarose (Sigma) and rocking for 2 h at 4 °C. The resin was then isolated by centrifugation at 1000 × g, rinsed three times with lysis buffer, and protein complexes were eluted by incubation with 3xFLAG peptide (125 μg/mL in lysis buffer).
3
Real-time ChIP Analysis Protocol
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Real-time PCR-based ChIP analysis was performed as described previously [69 (link)]. Cells were incubated with medium containing 0.9% formaldehyde for 10 min at room temperature. Sonicated chromatin fragments averaged ~300 to 500 bp. Soluble chromatin was incubated with the M2 Anti-FLAG Agarose (Sigma). DNA was quantitated by real-time PCR. The amounts of products were determined relative to input chromatin. The primers used are listed in S2 Table .
4
Recombinant Kinetochore Protein Expression
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Open reading frames encoding kinetochore subunits were amplified from yeast genomic DNA and cloned into the pFL vector for insect cell expression. Generation of recombinant viruses expressing multiple subunits was performed according to the multi-Bac system (Trowitzsch et al., 2010 (link)). For expression and purification, insect cells were opened with a Dounce homogenizer in Lysis buffer (20 mM Na2HPO4/NaH2PO4, pH 7.0, 300 mM NaCl, and 1 mM EDTA) with Complete EDTA-free protease inhibitors (Roche). Cleared extracts were incubated with M2 anti-Flag agarose (Sigma-Aldrich) for 2 h, washed 3× with buffer, and eluted with 2 mg/ml 3×Flag peptide in Lysis buffer.
For purification of 6×His-tagged proteins, insect cells were lysed in 20 mM Na2HPO4/NaH2PO4, pH 7.0, 300 mM NaCl, 20 mM imidazole, 1 mM PMSF, and Complete protease inhibitors. Cleared extracts were incubated with Ni-NTA agarose (QIAGEN) for 2 h and washed three times, and proteins were eluted with 200 mM imidazole in Lysis buffer.
For purification of 6×His-tagged proteins, insect cells were lysed in 20 mM Na2HPO4/NaH2PO4, pH 7.0, 300 mM NaCl, 20 mM imidazole, 1 mM PMSF, and Complete protease inhibitors. Cleared extracts were incubated with Ni-NTA agarose (QIAGEN) for 2 h and washed three times, and proteins were eluted with 200 mM imidazole in Lysis buffer.
5
Protein Immunoprecipitation and Pull-Down
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Protein extracts were prepared in lysis buffer as previously described (von Morgen et al, 2017 (link)). For IPs and pull-downs, 2.5 mg of the whole-cell extract was incubated for 2 h at 4°C with 15 μl of M2 anti-FLAG agarose (Sigma-Aldrich), GFP-Trap agarose (ChromoTek), or protein G agarose (Cell Signaling) bound with primary antibodies. Beads were pelleted and washed three times in 20x bed volume of the lysis buffer. For immunoprecipitation, bound proteins were eluted by boiling in 2x LSB (Laemmli sample buffer) for 5 min. Inputs represent 5% of the extracts used for IP.
6
Comprehensive Immunoblot and Immunofluorescence Antibody Panel
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Primary antibodies to γ-H2AX (No. 2577; WB 1:1000; IF 1:200), Lamin A/C (No. 4777; WB 1:1000), BRCA1 (No. 9010; WB 1:1000; IF 1:200), K48-linkage specific polyubiquitin (No. 8081; WB 1:1000), FLAG-tag (No. 8146; WB 1:1000; IF 1:200), Myc-tag (No. 2276; WB 1:1000), GAPDH (No. 2118; WB 1:1000), Ku80 (No. 2753, WB 1:1000), ORC2 (No. 4736, WB 1:1000), Actin (No. 3700, WB 1:1000) and HRP-linked secondary antibody to rabbit IgG (No. 7074; WB 1:1000), mouse IgG (No. 7076; WB 1:1000) from Cell Signal Technology (CST, Danvers, MA, USA); antibody to β-tubulin (No. T0023; WB 1:1000) from Affinity Biologicals; antibody to HA (No. ab18181; WB 1:1000; IP 1:1000), RAD51 (No. ab63801; WB 1:1000; IF 1:200) from Abcam; BAP1 (No. sc-28283; WB 1:500) from Santa Cruz Biotechnology; primary antibody to Biotin (No. bs-0311R; WB 1:1000) from Bioss (Beijing, China); primary antibody to BARD1 (No. PA5-85707; WB 1:1000) and fluorogenic secondary antibody goat anti-rabbit Alexa Fluro-488, anti-rabbit Alexa Fluro-594, goat anti-mouse Alexa Fluro-488, anti-mouse Alexa Fluro-594 (No. A11034, A11037, A32723, A11032, respectively; IF 1:1000) from Invitrogen (Carlsbad, CA, USA); M2 anti-Flag agarose (No. A2220) from Sigma–Aldrich.
7
Immunoprecipitation of FLAG-tagged Proteins
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Immunoprecipitations (IPs) were performed on nuclear protein lysates prepared in low salt buffer containing protease inhibitors (150 mM NaCl, 50 mM TRIS pH 8.0, 1 mM EDTA, 1% NP40, 1 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mM PMSF). IPs of FLAG tagged proteins were performed using M2 anti- FLAG agarose and mouse IgG agarose (Sigma) overnight at 4 °C. Elution of FLAG tagged proteins was performed at 4 °C using 250 ug/ml of 3xFLAG peptide (Sigma) in 0.05% NP40 with horizontal shaking. Eluted protein fractions were separated by SDSPAGE and analyzed by western blot or liquid chromatography mass spectrometry.
8
Purification of PI4KIIIα protein complexes
9
Cell Transfection and Protein Analysis
10
HMGA1 Interactome Characterization
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Immunoprecipitations of HMGA1-FLAG fusions were performed from nuclear extracts of transiently transfected HEK293 cells using M2 anti-FLAG agarose (Sigma) as recommended by the manufacturer. Endogenous HMGA1 was immunopurified from nuclear extracts using a specific anti-HMGA1 antibody (Abcam). Immunopurifications with boiled anti-HMGA1 IgG were used as a control. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotting were performed as described previously (22 (link)). Antibodies used in western blot analyses were M2 anti-FLAG antibody (Sigma), anti-HMGA1 antibody (Abcam), anti-CTIP2 antibody (Abcam), anti-Cdk9 antibody (Pierce), anti-CycT1 antibody (Proteintech), anti-HEXIM1 antibody (Proteintech) and anti-LARP7 antibody (Proteintech) with the corresponding secondary horseradish peroxidase-coupled antibodies (Sigma) as recommended by the manufacturer.
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