The pFastBac-Flag-CaVAb was used as the construct for producing homotetrameric model voltage-gated Ca2+ channel19 (link). I199S, W195Y, and T206S constructs were generated via site-directed mutagenesis using QuickChange (Stratagene). Recombinant baculovirus were produced using the Bac-to-Bac system (Invitrogen), and T. ni insect cells were infected for large-scale protein purification. Cells were harvested 72 h post-infection and re-suspended in buffer A (50 mM Tris-HCl, pH = 8.0, 200 mM NaCl) supplemented with protease inhibitors and DNase. After sonication, digitonin (EMD Biosciences) was added to 1%, and solubilization was carried out for 1–2 h at 4 °C. Clarified supernatant was then incubated with anti-Flag M2-agarose resin (Sigma) for 1–2 h at 4 °C with gentle mixing. Flag-resin was washed with ten column volumes of buffer B (buffer A supplemented with 0.12% digitonin) and eluted with buffer B supplemented with 0.1 mg ml−1 Flag peptide. The eluant was concentrated and then passed over a Superdex 200 column (GE Healthcare) in 10 mM Tris-HCl pH =8.0, 100 mM NaCl and 0.12% digitonin. The peak fractions were concentrated using a Vivaspin 30K centrifugal device.
Anti flag m2 agarose resin
Manufactured by Merck Group
Sourced in United States
The Anti-Flag M2-agarose resin is a lab equipment product that is used for the purification of recombinant proteins expressing the FLAG tag. It is a pre-packed agarose resin that can be used in affinity chromatography to capture and purify FLAG-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Flag peptide, by Merck Group (3 mentions)
Ab42080, by Abcam (2 mentions)
Sc 47778, by SCBT (2 mentions)
To 12 gradient gels, by Thermo Fisher Scientific (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Odyssey imaging system, by LI COR (2 mentions)
Dca protein assay kit, by Bio-Rad (2 mentions)
Pierce ldh cytotoxicity assay kit, by Thermo Fisher Scientific (2 mentions)
Digitonin, by GE Healthcare (1 mentions)
Bac to bac system, by Thermo Fisher Scientific (1 mentions)
Superdex 200 column, by GE Healthcare (1 mentions)
Protein a g agarose beads, by Thermo Fisher Scientific (1 mentions)
Stripping buffer, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence kit, by GE Healthcare (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Gel pro analyzer, by Media Cybernetics (1 mentions)
Complete edta free, by Roche (1 mentions)
Pierce micro spin columns, by Thermo Fisher Scientific (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Anti flag m2 magnetic resin, by Merck Group (1 mentions)
21 protocols using anti flag m2 agarose resin
1
Purification of Recombinant Voltage-gated Ca2+ Channels
2
Endogenous Protein Co-Immunoprecipitation
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The co-immunoprecipitation (IP) of endogenous proteins from PC-3 whole cell extract was carried out using RAD51C antibody (Novus Biologicals, 2H11/6) or control IgG. The immunocomplex was bound to protein A/G agarose beads (20421, Thermo Scientific Pierce) to detect the bound ALKBH3 using western blotting. The co-IP experiments with overexpressed proteins were carried out by Flag-RAD51C complex pull down. Whole-cell extract was prepared by resuspension of the cells in hypotonic buffer (20 mM HEPES pH 7.4, 10 mM KCl and 1 mM MgCl2) containing protease inhibitors and passing the extract through a 25-gauge needle several times to induce osmotic lysis. Next, KCl was adjusted to 200 mM and Triton X-100 was added to final concentration of 1% and incubated on ice for 30 min. After removing the insoluble materials by centrifugation, the extract was then incubated with Anti-Flag M2-agarose resin (Sigma) for 4 h at 4°C and centrifuged. The supernatant was harvested and considered as the unbound fraction. The beads were washed extensively with PBS and bound material was eluted using SDS loading buffer.
3
Immunoprecipitation and Kinase Assays in HeLa Cells
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Immunoprecipitations from HeLa cells were done as described previously (Egea et al., 2005 (link)). Cell lysates were cleared by centrifugation and equal amounts were incubated with 40 µl of anti-FLAG M2-Agarose resin (Sigma-Aldrich) overnight at 4°C, washed four times with lysis buffer, and then analyzed by Western blot. Membranes were incubated overnight with the respective primary antibody, followed by incubation with the species-specific secondary HRP-coupled antibody, and proteins were detected with an enhanced chemiluminescence kit (GE Healthcare). For reblotting, membranes were stripped for 15 min with stripping buffer (Thermo Fisher Scientific) at RT, again blocked with BSA, and reblotted using a primary antibody.
In the kinase-activity assay, coexpressed kinase substrate GST-JMA4 was pulled down with glutathione sepharose 4B (GE Healthcare), and then eluted with loading buffer and subjected to SDS-PAGE for Western blotting. Quantifications of unsaturated Western blots were done using Gel-Pro Analyzer software (Media Cybernetics) normalizing to controls.
In the kinase-activity assay, coexpressed kinase substrate GST-JMA4 was pulled down with glutathione sepharose 4B (GE Healthcare), and then eluted with loading buffer and subjected to SDS-PAGE for Western blotting. Quantifications of unsaturated Western blots were done using Gel-Pro Analyzer software (Media Cybernetics) normalizing to controls.
4
NMNAT-1 Immunoprecipitation from 3T3-L1 Cells
5
Purification of Arabidopsis DXPS Enzyme
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A. thaliana DXPS (UniProt code Q38854) (59-717aa) were cloned into pETDuet-1 vector to generate a C-terminal FLAG tag on DXPS. DXPS were overexpressed in E. coli BL21(DE3) Rosetta 2 and cells were grown at 37 °C until OD600 of 0.6–0.8 was reached. Proteins were induced by addition of 1 mM IPTG and the cells were grown overnight at 18 °C. Cells were harvested at 7000g for 10 min, resuspended with lysis buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, and 10% (v/v) glycerol) supplemented with protease inhibitor (cOmplete EDTA-free; Roche), and sonicated. The cell lysate was clarified by centrifugation at 20,000g for 1 h at 4 °C and applied to Anti-FLAG M2 agarose resin (Sigma). The column was then washed with 5 CV buffer containing 50 mM Tris-HCl, pH 8.0, 300 mM NaCl, 5 mM ATP, 20 mM MgCl2 and 10% (v/v) glycerol followed by 5 CV buffer washing containing 50 mM Tris-HCl, pH 8.0, 500 mM NaCl and 10% (v/v) glycerol and 15 CV lysis buffer washing. Protein was eluted with lysis buffer supplemented with 0.1 mg/mL 3x FLAG peptides. Pure fractions were pooled, concentrated by 50 kDa cut-off Centrifugal Filter Unit (Amicon), flash frozen in liquid nitrogen and stored at −80 °C until use.
6
NLRP1 Mutation Immunoprecipitation Protocol
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A total of 2.5 × 105 HEK293T cells were transfected with 500 ng of WT or mutant NLRP1 (IRES-GFP). Eighteen hours posttransfection, cells were washed once with 1×DPBS and harvested in NP40 lysis buffer (1% NP40 (vol/vol), 10% glycerol (vol/vol), 20 nM Tris-HCl, 150 mM NaCl, 1 mM ethyleneglycol-bis-(β-aminoethylether)-N,N,N',N'-tetraacetic acid, 10 mM NaPPi, 5 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride) freshly supplemented with 1× cOmplete protease inhibitor cocktail (Roche, Basel, Switzerland). After lysing cells for 20 minutes on ice, cell debris was spun down and the supernatant was collected. Immunoprecipitation was performed using anti–FLAG-M2-agarose resin (Sigma) for 4 hours or overnight at 4°C. Beads were washed 3 times with lysis buffer before elution by boiling in SDS sample buffer for 10 minutes. Immunoblots were prepared using 4% to 12% gradient gels (Novex, Invitrogen, Carlsbad, Calif) and subsequently transferred to a PVDF membrane. Membranes were blocked in PBS/tween 20 with 5% skim milk for 60 minutes at room temperature and probed overnight at 4°C. The following antibodies were used: aNLRP1: AL176 (AdipoGen, San Diego, Calif), aDPP9: ab42080 (Abcam, Cambridge, UK), aFLAG: 9H1 (in-house), and aActin: sc47778 (SCBT).
7
Affinity Purification of FLAG-Tagged Proteins
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HEK 293T cells were seeded in 6-well plates at 5 × 105 cells/well in DMEM for 24 h. The cells were then transiently transfected with the indicated constructs. After 48 h, the cells were harvested and lysed by sonication. Lysates were clarified by centrifugation at 21,000 x g for 5 minutes. The soluble fractions were then normalized using the DC Protein Assay (BioRad). 100 μL of the lysates were combined with 100 μL of 2x sample loading buffer and incubated at 95 °C for 10 minutes. Equal protein amounts of the remaining sample lysates were loaded onto Pierce Micro-Spin Columns (Thermo Scientific) containing 100 μL of anti-FLAG-M2 agarose resin (Sigma) and the samples were rotated end-over-end at 4°C overnight. The samples were then washed 3x with 1 column volume (350 μL) of PBS. Proteins were eluted by rotating the resin at room temperature for 1 hour in 100 μL of PBS containing 150 ng/ μL 3×-FLAG peptide (Sigma Aldrich). 100 μL of 2x sample loading buffer was added to the eluate and samples were boiled at 95 °C for 10 minutes. Both lysates and eluates were analyzed by immunoblotting
8
Nampt/PBEF Protein Expression Analysis
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Cells were lysed in a buffer containing 10 mM Tris pH 8.0, 20 mM KCl, 0.5 mM EDTA, 200 mM NaCl, 0.1% Triton ×100. Identical amount of cell lysates proceeded SDS-PAGE and then immunoblot analysis by anti-Nampt/PBEF antibody (C20), anti-GADPH (1D4), anti-β-actin (C4) (Santa Cruz Biotech, CA, USA), and anti-caspase 9 (N2C3) (GeneTex, CA, USA) antibodies. The antibody for caspase 9 only detects the intact (inactive) form of enzyme. The cell culture medium from two 10 cm cell culture dishes was used for immunoprecipitation by anti-FLAG (M2) agarose resin (Sigma-Aldrich, MO, USA) and then conducted immunoblot analysis.
9
Affinity-based Protein Purification
10
Quantifying Cellular Cytotoxicity and Protein Interactions
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HEK293T cells were transiently transfected and inhibitor treated as indicated. OCI-AML2 and MV-4-11 cells were plated in 6-well culture plates at 1.0 × 106 cells/well and treated with VbP as indicated. Supernatants were analyzed for LDH levels using the Pierce LDH Cytotoxicity Assay Kit (Life Technologies). LDH levels were quantified relative to a lysis control where cells were lysed in 80 μL of a 9% Triton X-100 solution. For FLAG immunoprecipitation, cell pellets were lysed in Tris-Buffered Saline (TBS) with 0.5% NP-40 on ice for 20 min and centrifuged at 20000 x g for 10 min. The soluble fraction was retained, and protein concentrations were normalized to 1 mg/mL using the DCA Protein Assay kit (Bio-Rad). Lysates were incubated with 40 μL of anti-FLAG-M2 agarose resin (Sigma) overnight at 4°C. After washing 3 × 1 mL with PBS, bound proteins were eluted by incubating resin with 100 μL of PBS containing 150 ng/ μL 3 × -FLAG peptide for 1 h at 4°C. An equal volume of 2 × sample loading dye was added to the eluate and incubated at 95°C for 10 min. For immunoblotting, cells were washed 2 × in PBS (pH = 7.4), resuspended in PBS, and lysed by sonication. Protein concentrations were determined and normalized using the DCA Protein Assay kit (Bio-Rad). The samples were separated by SDS-PAGE, immunoblotted, and visualized using the Odyssey Imaging System (Li-Cor).
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