Rat anti flag antibody

Manufactured by BioLegend

The Rat anti-FLAG antibody is a monoclonal antibody that recognizes the FLAG epitope, a short peptide tag that can be fused to recombinant proteins. The antibody can be used to detect and purify FLAG-tagged proteins in various applications, such as immunoprecipitation, Western blotting, and immunoaffinity chromatography.

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Lab products found in correlation

12 mm round glass coverslips, by Thermo Fisher Scientific (2 mentions) Surebeads protein g magnetic beads, by Bio-Rad (1 mentions) Endo h, by New England Biolabs (1 mentions) Pngase f, by New England Biolabs (1 mentions) Goat α rat igg cy2, by Jackson ImmunoResearch (1 mentions) Goat α mouse igg alexa657, by Thermo Fisher Scientific (1 mentions) Application suite x software, by Leica (1 mentions) Mouse α pdi, by Thermo Fisher Scientific (1 mentions) Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

Anti-FLAG (10 citations) Rat anti-FLAG (6 citations) Anti-FLAG antibody (5 citations) Rat anti-FLAG antibody L5 (2 citations)

4 protocols using rat anti flag antibody

Characterization of NADPH oxidase complex

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Strep tagged-EROS was co-transfected in HEK293-F cells with GFP-gp91phox and p22phox. Then, 48 hr post-transfection cells were harvested and cell membranes were prepared as described in Ceccon et al., 2017 (link). Membranes were solubilised with 1% LMNG in 50 mM HEPES pH 7.5, 100 mM NaCl, 20% (v/v) glycerol, and passed through Streptactin-resin. Bound proteins were eluted from the column with 5 mM desthiobiotin in 50 mM HEPES pH 7.5, 100 mM NaCl, 5% (v/v) glycerol, and then subjected to SEC using a Superose 6 30/150 column.
Lysates from 3.10⁷ to 4.10⁷ of cells were subjected to immunoprecipitation according to the manufacturer’s protocol for the Sure Beads Protein G Magnetic Beads (161-4023, Bio-Rad) using a rat anti-FLAG antibody (BioLegend), the mouse anti-gp91phox antibody (Santa Cruz), and an IgG2aK isotype control antibody (14-4321-82, Invitrogen). Eluates from FLAG, gp91phox, and IgG control beads were analysed by Western blotting. Where indicated, lysates were treated with PNGase F or EndoH (New England Biolabs) following the manufacturer’s recommendation.

Randzavola L.O., Mortimer P.M., Garside E., Dufficy E.R., Schejtman A., Roumelioti G., Yu L., Pardo M., Spirohn K., Tolley C., Brandt C., Harcourt K., Nichols E., Nahorski M., Woods G., Williamson J.C., Suresh S., Sowerby J.M., Matsumoto M., Santos C.X., Kiar C.S., Mukhopadhyay S., Rae W.M., Dougan G.J., Grainger J., Lehner P.J., Calderwood M.A., Choudhary J., Clare S., Speak A., Santilli G., Bateman A., Smith K.G., Magnani F, & Thomas D.C. (2022). EROS is a selective chaperone regulating the phagocyte NADPH oxidase and purinergic signalling. eLife, 11, e76387.

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Visualizing BiP-3xFLAG in HEK293 Cells

Cited 1 time

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BiP-3xFLAG HEK293 cells (0.12 × 106) were plated on 12-mm round glass coverslips (Thermo Fisher Scientific) coated with 0.15 mg/ml poly-lysine in 24-well plates. The following day, cells were fixed and immunostained using a standard procedure (Sundaram et al., 2017 (link)). Rat anti-FLAG antibody (#637303; Biolegend) and mouse α-PDI (#MA3-018; Affinity Bioreagents) were used as primary antibodies. Goat α-RAT IgG-Cy2 (#112-225-167; Jackson ImmunoResearch) and Goat α-Mouse IgG-Alexa657 (#A-21235; Invitrogen) were used as secondary antibodies. Cells were imaged on Leica scanning confocal consisting of an inverted microscope (Leica SP6/SP8) and an HC PL APO 63×/1.4 (CS2 No: 11506350) oil objective lens (Leica) and was controlled by the Leica Application Suite X software. Sequential image scanning at 1× zoom, 100 Hz, 1,024 × 1,024 pixels, and with line averaging set at four was used to collect images for displayed images.

Sun S., Li X, & Mariappan M. (2022). Signal sequences encode information for protein folding in the endoplasmic reticulum. The Journal of Cell Biology, 222(1), e202203070.

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Anx Protein Binding Assay

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PIP strip membranes (Invitrogen) were blocked in blocking buffer (TBS: 20 mM Tris pH 8.0 + 150 mM NaCl, with 0.1% Tween-20 and 1% non-fat milk) for one hour at RT and incubated with purified Anx proteins (0.5 µg/ml) and 100 µM CaCl₂ or 100 µM EGTA for one hour at RT. The membranes were washed three times with TBS-T (TBS with 0.1% Tween-20) and bound Anxs were detected by immunoblotting using Rat anti-FLAG antibody (1:5000; Biolegend) and ECL (ThermoFisher).

Nakamura M, & Parkhurst S.M. (2023). Calcium influx rapidly establishes distinct spatial recruitments of Annexins to cell wounds. bioRxiv.

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Immunofluorescence Imaging of BiP-3xFLAG HEK293 Cells

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BiP-3xFLAG HEK293 cells (0.12 X 10 6 ) were plated on 12 mm round glass coverslips (Fisher Scientific) coated with 0.15 mg/mL poly-lysine in 24-well plates. The following day, cells were fixed and immunostained as previously described (Sundaram et al., 2017) . Rat anti-FLAG antibody (BioLegend) and mouse anti-PDI antibody (Affinity Bioreagents) were used as primary antibodies. Goat anti-mouse Alexa 647 and Goat anti-rat Cy2 (Jackson Immuno Research) were used as secondary antibodies. The cells were imaged on a Leica scanning confocal microscope as previously described (Sundaram et al., 2017) .

Sun S., Li X., & Mariappan M. (2020). Signal sequences encode information for protein folding in the endoplasmic reticulum.

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