The VvWRKY5 CDS was inserted into the pHBT-AvrRpm1-HA vector (VvWRKY5-HA), whereas the VvJAZ2 CDS was ligated into a pHBT-AvrRpm1-FLAG vector (VvJAZ2-FLAG). These vectors were transiently inserted into protoplasts derived from ‘Gamay’ grape calli and then incubated for 6 h. Anti-FLAG-agarose beads (Abmart, Shanghai, China) were used for protein purification. The IP buffer was used to wash the beads, whilst anti-FLAG and anti-HA antibodies (Abmart, Shanghai, China) were used to detect the proteins by western blotting. Supplementary Data Table S3 shows the details of all primers used here.
Anti flag agarose beads
Manufactured by Abmart
Sourced in China
Anti-FLAG-agarose beads are a resin-based affinity matrix designed for the purification of proteins tagged with the FLAG epitope. The beads are composed of agarose and covalently linked with anti-FLAG monoclonal antibodies, allowing for the efficient capture and isolation of FLAG-tagged proteins from various biological samples.
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Lab products found in correlation
3 protocols using anti flag agarose beads
1
VvWRKY5 and VvJAZ2 Protein Interaction
2
Protein Interaction Analysis via Affinity Purification
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Recombinant constructs GFP-OsCBSX3 and Myc-OsDRAP1 were introduced into tobacco leaves as the above protocol in dual-LUC or BiFC assays, respectively, and protein extracts were prepared as described by Sawa et al. (2007 (link)). The protein extracts were precipitated with anti-Flag agarose beads (Abmart, http://www.ab-mart.com/ ) or anti-Myc agarose beads (CMC Scientific, http://www.cmcscientific.com ) overnight. Then proteins bound to beads were resolved by SDS-PAGE and detected with Western blot using anti-GFP antibody (Sigma), and anti-Myc antibody (MBL, http://www.mblintl.com/ ).
3
FLAG-Mediated Protein Immunoprecipitation
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Agrobacteria harboring the corresponding constructs were co-infiltrated into N. benthamiana leaves. The infiltrated leaves were harvested 3 d postinfiltration, ground in liquid nitrogen, and resuspended with IP buffer (25 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% v/v Nonidet P-40, 5% v/v glycerol, 1 mM PMSF, 20 μM MG132, 5 mM DTT, and 1× protease inhibitor cocktail). After centrifugation at 12,000 g for 15 min at 4°C, 20 µL of anti-FLAG agarose beads (Abmart, Shanghai, China) were added and incubated for 3 h at 4°C. The beads were washed 3 times with wash buffer (10 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.5 mM EDTA, 0.5% v/v Triton X-100, 0.5 mM DTT, 1 mM PMSF, and 1× protease inhibitor cocktail). Immunoprecipitates were analyzed by western blot.
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Agrobacteria harboring the corresponding constructs were co-infiltrated into N. benthamiana leaves. The infiltrated leaves were harvested 3 d postinfiltration, ground in liquid nitrogen, and resuspended with IP buffer (25 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% v/v Nonidet P-40, 5% v/v glycerol, 1 mM PMSF, 20 μM MG132, 5 mM DTT, and 1× protease inhibitor cocktail). After centrifugation at 12,000 g for 15 min at 4°C, 20 µL of anti-FLAG agarose beads (Abmart, Shanghai, China) were added and incubated for 3 h at 4°C. The beads were washed 3 times with wash buffer (10 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.5 mM EDTA, 0.5% v/v Triton X-100, 0.5 mM DTT, 1 mM PMSF, and 1× protease inhibitor cocktail). Immunoprecipitates were analyzed by western blot.
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