Immunoblotting was conducted similarly to those published previously (32 (link), 33 (link)). Briefly, cells were washed once with pre-warmed 1X PBS and then lysed directly with ice-cold lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% (v/v) NP-40, 0.5% sodium-deoxycholate, and Complete™ protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Whole cell lysates (50 μg) were resolved on a 10% SDS-PAGE (BioRad, Hercules, CA) and transferred to a nitrocellulose membrane (Santa Cruz Biotechnology, Inc., Dallas, TX). The membrane was probed with the following antibodies: mouse polyclonal anti-FLAG antibody (1:4000 dilution), mouse monoclonal anti-β-actin antibody (1:5000 dilution) (Sigma Aldrich, St. Louis, MO), and mouse monoclonal anti-GAPDH antibody (1:5000 dilution) (Santa Cruz Biotechnology, Inc., Dallas, TX). The nitrocellulose membrane was then probed with an anti-mouse HRP-conjugated secondary antibody (Cell Signaling, Danvers, MA). The signal was detected using Supersignal West Dura (Pierce, Rockford, IL) with a BioRad ChemiDoc XRS imaging system (BioRad, Hercules, CA). All primary and secondary antibodies were diluted in 1X TBST. Expression of protein and loading control densitometry analysis was performed using Image Lab v4.1 software (BioRad, Hercules, CA).
Mouse polyclonal anti flag antibody
Manufactured by Merck Group
Sourced in United States
The Mouse polyclonal anti-FLAG antibody is a laboratory reagent used for the detection and purification of proteins tagged with the FLAG peptide sequence. It is produced by immunizing mice with the FLAG peptide and collecting the resulting polyclonal antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Santa Cruz Biotechnology (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Mouse monoclonal anti β actin antibody, by Merck Group (1 mentions)
Mouse anti gapdh monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Anti mouse hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Image lab v4, by Bio-Rad (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Cfx384 real time system, by Bio-Rad (1 mentions)
Amersham ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Hrp conjugated anti mouse igg antibody, by Bio-Rad (1 mentions)
Fura 2 am, by Thermo Fisher Scientific (1 mentions)
4 protocols using mouse polyclonal anti flag antibody
1
Western Blot Analysis Protocol
2
Chromatin Immunoprecipitation of IAA8 Transcription Factor
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Ten-day-old iaa8-1 and iaa8-1/IAA8 OX seedlings were treated with cold (4°C) and H2O2 for 12 h. Chromatin immunoprecipitation (ChIP) was carried out as described (Gendrel et al., 2002 (link)) using mouse polyclonal anti-flag antibody (1:3000; Sigma, USA). PCR amplification was performed quantitatively using the CFX384 Real-Time System (Bio-Rad, USA). The immunoprecipitation was replicated three times. The ChIP primers are listed in Supplementary Table S2
3
SDS-PAGE Analysis of Cell Fractions
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SDS-PAGE of cell fractions was performed according to the method described by Laemmli (1970) [39 (link)]. Cell fraction samples were prepared by incubation at 100 °C for 5 min and separated on 12.5% polyacrylamide gels. Proteins were transferred to Amersham Hybond™-N+ nylon hybridization transfer membranes (GE Healthcare) at 450 mA for 40 min in a semi-dry transfer and then probed with mouse polyclonal anti-FLAG antibody (Sigma-Aldrich). The membranes were then incubated with rabbit anti-mouse antibody coupled to horseradish peroxidase. The presence of LdtP was detected by chemiluminescence using the Amersham ECL™ western blotting detection reagents (GE Healthcare) according to the manufacturer’s instructions.
4
Biochemical Regulation of STIM1/Orai1 Signaling
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Thr, TG (thapsigargin; a nonreversible antagonist of the SERCA [sarcoendoplasmic Ca 2+ -ATPase pump]), ionomycin (Iono; Ca 2+ protonophore), calpeptin (inhibitor of CAPN1 [calpain1]), N-(2ʹ,5ʹ-dimethoxy-[1,1ʹ-biphenyl]-4-yl)-3fluoroisonicotinamide; Orai1 specific blocker (Synta66), anti-Orai1 antibody, mouse polyclonal anti-flag antibody and rabbit polyclonal anti-β-actin antibody, APY (apyrase) and aspirin were from Sigma-Aldrich. Fura-2/AM was purchased from Molecular probes. Mouse monoclonal anti-STIM1 antibody (catalog no. 610954; anti-N-STIM1 [anti-Nterminal-STIM1 antibody] antibody, clone 44 GOK-1, epitope: amino acids 25-139 of human STIM1) and the antibody raised toward the C-terminal domain of STIM1 (anti-C-terminal-STIM1 antibody) were supplied by Merck Millipore; meanwhile, specific anti-STIM1B (540) antibody was generated in the Dr Niemeyer's laboratory (Universität des Saarlandes, Saarbrücken, Germany). HRP-conjugated anti-mouse and anti-rabbit IgG antibodies were from Jackson Immunoresearch and, alternatively, in some experiments an HRP-conjugated anti-mouse IgG antibody from Biorad was used. Overexpression plasmids: human SARAF pIRES2-eGFP-RV was generated in our laboratory, 24 (link) and rabbit polyclonal anti-SARAF antibody (epitope: aminoacids 33-62 of the N-terminal region of human SARAF; cat. no. PA5-24237), qRT-PCR primers for STIM1B (540) 25 (link) and
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