FLAG-tagged CMG (5 nM final concentration) was incubated in 31 μL of nucleoplasmic extract (NPE) or IP buffer (1× ELB salts [2.5 mM MgCl2, 50 mM KCl, 10 mM HEPES at pH 7.7], 0.1% NP-40, 0.1 mg/mL BSA) in the presence of 200 μM p97-i (Sigma NMS-873) for 30 min at room temperature. Fifteen microliters of each reaction was added to 5 μL of anti-FLAG resin (Sigma) and incubated with end-over-end rotation for 1.5 h at 4°C. The beads were washed five times with 80 vol of IP buffer. Samples were eluted from anti-FLAG resin with 3 vol of IP buffer + 0.2 mg/mL 3xFLAG peptide prior to mixing with 1 vol of 2× SDS sample buffer. Samples were resolved by SDS-PAGE and immunoblotted as described above. All immunoprecipitation experiments were performed at least twice, with the exception of Figure 4 B, which was performed once.
Anti flag resin
Manufactured by Merck Group
Sourced in United States
Anti-FLAG resin is a laboratory tool used for the purification and detection of proteins tagged with the FLAG epitope. It consists of agarose beads coated with an anti-FLAG antibody, which specifically binds to the FLAG tag on the target proteins, allowing their separation and isolation from complex samples.
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Lab products found in correlation
Flag peptide, by Merck Group (5 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Hitrap q hp column, by GE Healthcare (2 mentions)
Amicon ultra 0.5 10 kda cutoffspin filters, by Merck Group (2 mentions)
Silverquest staining kit, by Thermo Fisher Scientific (2 mentions)
Protein a g bead, by Santa Cruz Biotechnology (2 mentions)
Streptavidin hrp, by Vector Laboratories (1 mentions)
E pha, by Vector Laboratories (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Cellytic mt cell lysis reagent, by Merck Group (1 mentions)
Superdex 200 column, by GE Healthcare (1 mentions)
Complete edta free protease inhibitor, by Roche (1 mentions)
Glutathione agarose beads, by Merck Group (1 mentions)
Complete protease inhibitor cocktail tablet, by Roche (1 mentions)
Trypsin gold, by Promega (1 mentions)
Igepal ca 630, by Merck Group (1 mentions)
Rabbit igg agarose resin, by Merck Group (1 mentions)
Chaps, by Merck Group (1 mentions)
Vegfr2 55b11, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Roche (1 mentions)
30 protocols using anti flag resin
1
FLAG-tagged CMG Immunoprecipitation Protocol
2
Purification and Analysis of Periostin
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Cell culture supernatant (50 mL) was collected from OVCAR3-PN, Pro5-PN, and Lec4-PN cells with the addition of protease inhibitors. Periostin was purified on anti-Flag resin (Sigma-Aldrich) according to the manufacturer instructions. Proteins were separated on NuPage 4–12% BisTris gel using 1X MES buffer prior to transfer to PVDF membrane. Blots were blocked in 3% BSA/1X TBST before detection of bisecting glycans using (1:5,000) dilution of biotin labeled E-PHA (Vector Labs) and (1:10,000) dilution of streptavidin HRP (Vector Labs) followed by enhanced chemiluminescent detection. The blot was stripped in Pierce (Thermo) stripping buffer, blocked in 5% nonfat milk 1XTBST and detected using (1:250) dilution of antibody to periostin (Santa Cruz Biotechnologies).
3
Immunoprecipitation of Ptf1a-Flag in MEFs
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MEFs infected with GFP or Ptf1a-Flag (flag-tagged Ptf1a) lentiviruses were collected at day 12 and lysed with the CelLytic MT Cell Lysis Reagent (Sigma, C3228) containing protease inhibitor cocktail (Roche), followed by centrifugation in a microfuge at 12,000 r.p.m. for 20 min. The protein concentration was determined by a standard bicinchoninic acid assay kit (Beyotime). Immunoprecipitation was carried out by incubating the cell lysates with anti-Flag resin (Sigma, A2220) at 4 °C overnight. After incubation, the resins were washed three times with Tris-buffered saline. The precipitates were incubated with 3 × Flag peptide elution solution with gentle shaking for 30 min at 4 °C. The eluates and whole-cell extracts (input) were separated on an 8% SDS-PAGE gel and electrotransferred to polyvinylidene fluoride membranes (Immobilon-P, Millipore). Western blotting was performed using the following primary antibodies: mouse anti-Rbpj (Santa Cruz, sc-271128, 1:500), mouse anti-Flag (Sigma, F1804, 1:5000), rabbit anti-GFP (MBL, 598, 1:2000), mouse anti-β-actin (Sigma, A5316, 1:5000), and secondary antibodies: goat anti-mouse or rabbit IgG horseradish peroxidase (KangChen, KC-MM-035, KC-RB-035, 1:5000). The membranes were incubated with enhanced chemofluorescent reagent (Pierce Biotechnology, 34095) and imaged with a digital imager (FluorChem E System, ProteinSimple).
4
Recombinant Myosin Preparation and Labeling
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Recombinant myosin was prepared as previously described (Hariadi et al., 2015 (link)). The construct contained (N to C terminus): the motor domain of myosin Va (residues 1–815; Gallus gallus), the lever arm of myosin VI (residues 917–992; Sus scrofa), a GCN4 leucine zipper (for dimerization), a SNAP tag (for Cy3 labeling), and a FLAG tag (for purification). Myosin was cloned in pBiex-1 (71234; EMD Millipore) and expressed through transient transfection in Sf9 insect cells using the Escort IV system (L3287; Sigma-Aldrich). Cells were lysed, and clarified lysate was incubated with anti-FLAG resin (Sigma-Aldrich) for 1 h at 4°C. Myosin bound to resin was then labeled with Cy3 DNA oligonucleotides overnight, washed, and eluted with FLAG peptides (Sigma-Aldrich). Labeled myosin was stored at −20°C in 20 mM imidazole, 150 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 55% (vol/vol) glycerol, 1 µg/ml PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin, pH 7.4.
5
In vitro SIRT1 Deacetylation Assay
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The in vitro SIRT1 deacetylation assay was performed as previously described [83 (link)]. Briefly, 500ng of the purified SIRT1-FLAG from HEK293F cells were incubated with 1200ng of hyperacetylated histones (purified by acid extraction) or 500ng of purified FLAG-p53 (anti-FLAG resin, Sigma-Aldrich) in 30μl of deacetylation buffer (50 mM-Tris pH7.8, 4mM MgCl2, 0.2mM DTT) for 15min at 37° C in the presence or absence of 1 mM NAD+ and/or SA at 0-10mM (dissolved in Tris 50mM pH 7.8). The reaction was stopped by adding Laemmli sample buffer. Deacetylation was monitored by quantification of H3K9ac/H3, H4K16ac/H4 and p53K382ac/p53 levels.
6
Purification of Cdt1-3FLAG Protein
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The baculoviruses for Cdt1-3FLAG expression were infected into Sf21 insect cells, and cultured at 27°C for 60 h. Cell lysate was prepared with 0.5 M NaCl–containing buffer B (50 mM Tris–HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 10% glycerol, 1× Protease inhibitor cocktail [Roche Applied Science], 1 mM PMSF, 2 μg/ml Leupeptin), and passed on Diethylaminoethyl (DEAE) column. The flowthrough fraction was mixed with anti-FLAG resin (Sigma-Aldrich) and Cdt1-3FLAG was eluted with 200 µg/ml 3× FLAG peptides (Sigma-Aldrich).
7
MHF1 Protein Immunoprecipitation in HeLa Cells
8
Purification of HDAC1/MIDEAS/DNTTIP1 Complexes
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HDAC1/MIDEAS/DNTTIP1 complexes were purified from 1.2 l of HEK293F cells. After sonication in a buffer containing 50 mM Tris/Cl pH 7.5, 100 mM potassium acetate, 10% (v/v) glycerol, 0.5% (v/v) Triton X-100 and Complete EDTA-free protease inhibitor (Roche) (buffer A), the insoluble material was removed by centrifugation. The complex was then bound to anti-FLAG resin (Sigma), washed twice with buffer A; three times with buffer B (50 mM Tris/Cl pH 7.5, 50 mM potassium acetate, 5% (v/v) glycerol, 0.5 mM TCEP); incubated with 0.5 mg RNaseA for 1 h at 4 °C and then washed five times with buffer B. TEV protease was then used to release the MiDAC complex from the resin overnight on a roller at 4 °C. The complex was gel filtrated on a Superdex-200 column (GE Healthcare) in 25 mM HEPES pH 7.5, 50 mM potassium acetate, 0.5 mM TCEP before making EM grids.
9
Recombinant Human Cardiac Myosin Purification
10
Investigating Protein-Protein Interactions
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HEK293T cells transiently expressing FLAG-tagged proteins were lysed in the lysis buffer [20 mM Tris-Cl (pH 7.5), 100 mM KCl, 1% NP-40, 1 mM EDTA, 10% glycerol, 10 mM sodium pyrophosphate, 1 mM PMSF, 3 mM DTT, and protease inhibitor cocktail (Calbiochem)]. 3 ml of cell lysates from ~ 2 × 107 cells were incubated with 3 ml of testis lysates from six 8-week mice for 2 h at 4°C. After the incubation, co-IP experiments were carried out with anti-FLAG resin (Sigma) as described previously [34 (link)].
GST-pull down experiments were performed as described before [34 (link)]. Briefly, bacterially expressed GST-BBS7 bound to glutathione-agarose beads (Sigma) were incubated respectively with bacterial lysate containing His-tagged luciferase, TTC25, TTC4, TTC9c, TTC36, or TTC39c for 2 h at 4°C. The beads were rinsed with wash buffer [20 mM Tris-Cl (pH 7.5), 150 mM KCl, 0.5% NP-40, 1 mM EDTA, 10% glycerol, 10 mM sodium pyrophosphate, 1 mM PMSF, and protease inhibitor cocktail (Calbiochem)] for three times. The samples were then analyzed by western blotting.
GST-pull down experiments were performed as described before [34 (link)]. Briefly, bacterially expressed GST-BBS7 bound to glutathione-agarose beads (Sigma) were incubated respectively with bacterial lysate containing His-tagged luciferase, TTC25, TTC4, TTC9c, TTC36, or TTC39c for 2 h at 4°C. The beads were rinsed with wash buffer [20 mM Tris-Cl (pH 7.5), 150 mM KCl, 0.5% NP-40, 1 mM EDTA, 10% glycerol, 10 mM sodium pyrophosphate, 1 mM PMSF, and protease inhibitor cocktail (Calbiochem)] for three times. The samples were then analyzed by western blotting.
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