Indicated cell lines were plated in 10 cm dishes and transfected with plasmids encoding the indicated proteins. Forty-eight hours after transfection, cells were harvested and lysed in lysis buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 0.5% NP40 (Igepal CA630); 2 mM EDTA; 1 mM DTT; 1U benzonase; protease inhibitors; in water). Lysates were clarified by centrifugation at 15 000 g and diluted to 2 mg/ml. Lysates were then pre-cleared with 30 μl of packed Protein G Agarose Fast Flow (Millipore) for 45 min at 4°C with end-over-end rotation. For FLAG immunoprecipitation experiments, pre-cleared lysates were incubated with 25 μl of packed FLAG M2 agarose beads (Sigma-Aldrich) (or Protein G Agarose Fast Flow beads as control) and incubated for 3 h at 4°C with end-over-end rotation. For V5 immunoprecipitation experiments, pre-cleared lysates were incubated with 2 μg of V5 antibody (Cell Signaling) (or no antibody as control) overnight at 4°C with end-over-end rotation. The following day, lysates were incubated with 30 μl packed Protein G Agarose Fast Flow (Millipore) for 2 h at 4°C with end-over-end rotation. For both FLAG and V5 IP experiments, beads were washed five times with 1 ml of cold lysis buffer and eluted by boiling beads in 40 μl 1× SDS-PAGE sample buffer. Input and immunoprecipitation samples were then analyzed by western blotting.
V5 antibody
Manufactured by Cell Signaling Technology
The V5 antibody is a laboratory reagent used for the detection and purification of proteins tagged with the V5 epitope. The V5 epitope is a small peptide sequence derived from the P and V proteins of the paramyxovirus SV5. The V5 antibody specifically binds to this epitope, allowing for the identification and isolation of V5-tagged proteins in various experimental applications.
Automatically generated - may contain errors
5 protocols using v5 antibody
1
Immunoprecipitation of Protein Complexes
2
Antibody Immunoblotting Protocol
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Pex30 antibody (Joshi et al., 2016 (link)) was a kind gift from William Prinz (National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD). Dpm1 antibody (mouse monoclonal 5C5A7; dilution 1:10,000) was purchased from Invitrogen. HA antibody (rat monoclonal 3F10; dilution 1:2,000) was purchased from Roche. V5 antibody (rabbit monoclonal D3H8Q; dilution 1:5,000) was purchased from Cell Signaling. MYC antibody (mouse monoclonal 9E10; dilution 1:1,000) was purchased from Roche. LD dye BODIPY493/503 was purchased from Invitrogen and used at a final concentration of 1 µg/ml. MDH was purchased from Abgent and used at 0.1 mM.
3
Immunogold Electron Microscopy Protocol
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Immunogold electron microscopy was performed as described previously (Zhang et al., 2004 (link)). V5 antibody (Cell Signaling Technology, 13202) was used in this experiment.
4
SARS-CoV-2 Spike Protein Interaction
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HEK293Ts were transfected using PEI with 0.5 μg pCG-SARS CoV2 Spike-V5 and 0.5 μg of pCG IFITM1, IFITM2, or IFITM3. 24 h later, samples were lysed with IP lysis buffer (50 mM, Tris pH8, 150 mM NaCl, 1 % NP40, protease inhibitor) for 10 min on ice. Lysed samples were centrifuged and incubated for 3 h with Pierce Protein A/G Magnetic beads (88802) which were pre-incubated overnight with V5 antibody (Cell signaling E9H80; 5 μg of primary antibody per 10 μl of beads per sample).
5
Immunoprecipitation of HER3 Mutants
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For immunoprecipitation (IP) experiments, stably transfected MCF10AHER2 cells expressing HER3 WT and mutant (G284R, D297Y, T355I and E1261A) were grown till 70% confluency and lysed in 1% NP40 lysis buffer containing 20 mM Tris, pH 7.4, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM EGTA, 5 mM NaPyro-PO4, 50 mM NaF, 10 mM β-glycero-PO4. 700 micrograms (µg) of cleared lysate was incubated with 2 µg of V5 antibody (Cell Signaling Technology, Cat# 13202) overnight at 4° C. The next day, 5 µl Protein G magnetic beads (Millipore) were added per 1 µg of antibody and incubated for 1 hr at 4° C. Resuspended beads were washed with lysis buffer and boiled in SDS-PAGE loading buffer. The levels of V5, p-Tyr and HER2 from immunoprecipitated samples were analyzed by western blotting.
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