Anti flag m1

Manufactured by Merck Group

Sourced in Denmark, Germany, Macao

The Anti-Flag M1 is a lab equipment product designed for use in molecular biology and biochemistry applications. It functions as an affinity tag for the purification and detection of recombinant proteins. The core purpose of this product is to enable researchers to isolate and analyze target proteins of interest.

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Lab products found in correlation

Irdye 680 and 800 nm, by LI COR (2 mentions) Anti bip, by BD (2 mentions) Bafilomycin a1, by Merck Group (2 mentions) Protein a sepharose beads, by Merck Group (2 mentions) Pe conjugated goat anti mouse secondary antibody, by BD (1 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions) Cytoflex s, by Beckman Coulter (1 mentions) Sc 66931, by SCBT (1 mentions) Sds loading buffer, by Bio-Rad (1 mentions) Chemidoc mp imager, by Bio-Rad (1 mentions) Thapsigargin, by MP Biomedicals (1 mentions) Anti α1 antitrypsin, by Agilent Technologies (1 mentions) Anti tubulin, by Merck Group (1 mentions) Dithiothreitol (dtt), by Merck Group (1 mentions) Anti ha, by Fortrea (1 mentions) Puromycin, by InvivoGen (1 mentions) Glutamic acid, by Merck Group (1 mentions) Biotin, by Merck Group (1 mentions) Anti v5, by Thermo Fisher Scientific (1 mentions) Trehalose, by Merck Group (1 mentions)

8 protocols using anti flag m1

Quantifying Cell Surface and Total CCR5 Expression

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WT and mutant CCR5 coding sequences were tagged with a FLAG sequence in N-terminal position as described in ref. ^{60 (link)}. HEK 293 cells were transfected with WT and mutant CCR5 using Lipofectamine 2000 (Life Technologies). Transfected cells were detached at day 2 and fixed in 4% paraformaldehyde for 10 min at room temperature (RT). The total amount of CCR5 in the cells was determined after permeabilization of fixed cells with 0.05% saponin in PBS with BSA 3% for 10 min at RT. Unpermeabilized cells were used to measure CCR5 surface expression. Cells were incubated with an antibody against the FLAG epitope (anti-Flag M1 used at 1:500, Sigma-Aldrich) followed by a PE-conjugated goat anti-mouse secondary antibody (used at 1:100, BD Biosciences). The stained cells were washed and analyzed on a Cytoflex S flow cytometer (Beckman Coulter) using the CytExpert Sofware v2.4.

Claireaux M., Robinot R., Kervevan J., Patgaonkar M., Staropoli I., Brelot A., Nouël A., Gellenoncourt S., Tang X., Héry M., Volant S., Perthame E., Avettand-Fenoël V., Buchrieser J., Cokelaer T., Bouchier C., Ma L., Boufassa F., Hendou S., Libri V., Hasan M., Zucman D., de Truchis P., Schwartz O., Lambotte O, & Chakrabarti L.A. (2022). Low CCR5 expression protects HIV-specific CD4+ T cells of elite controllers from viral entry. Nature Communications, 13, 521.

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Co-immunoprecipitation of FLAG-β2AR Complexes

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Co-immunoprecipitation assays was carried out with HEK293 cells homogenized in 0.4 mL of lysis buffer and centrifuged for 30 minutes at 4°C and 16,100 xg. Supernatants were transferred to fresh centrifuge tubes, and 10 μL Protein A-Sepharose beads (GE17–0780-01, Millipore, MA) and 1.0 μg of control IgG antibody (sc-66931, SCBT, CA) were added. Tubes were incubated for 1 hour at 4C to preclear samples. Precleared samples (1 mL) were incubated with 30 μL Protein A-Sepharose beads and 2 μg of anti-Flag M1 (Sigma-Aldrich, # F3040) or anti-IgG antibody (sc-66931, SCBT, CA) at 4°C overnight. After incubation, beads were rinsed with lysis buffer 3 times. The washed beads were mixed with 30 μL 2x SDS loading buffer (#161–0747, Bio-Rad Laboratories, CA), subjected to electrophoresis, and Western blotted for FLAG-β₂AR, Nb80, and PDE4D5. Gel images were taken and quantified using a Bio-Rad ChemiDoc MP Imager.

Martinez J.M., Shen A., Xu B., Jovanovic A., de Chabot J., Zhang J, & Xiang Y.K. (2023). Arrestin-dependent nuclear export of phosphodiesterase 4D promotes GPCR-induced nuclear cAMP signaling required for learning and memory. Science signaling, 16(778), eade3380.

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ER Stress Protein Regulation Analysis

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The following chemicals were used: thapsigargin (MP Biomedicals, Santa Ana, CA), tunicamycin and
kifunensine (Calbiochem, Billerica, MA), puromycin (InvivoGen, San Diego, CA), and bafilomycin A1,
MG-132, and DTT (Sigma-Aldrich, St. Louis, MO). For Western blot analyses, the following antibodies
were used: anti-FLAG M1 and anti-tubulin (Sigma-Aldrich), anti–α1-antitrypsin (Dako,
Glostrup, Denmark), anti–14-3-3z and anti-SEL1L (Santa Cruz Biotechnology, Dallas, TX),
anti–S-tag and anti–OS-9 (Abcam, Cambridge, England), anti–GRP94 9G10 and
anti-KDEL (Enzo, Farmingdale, NY), anti-GRP94 (provided by Ineke Braakman, Utrecht University,
Utrecht, Netherlands), anti-HA (Covance, Princeton, NJ), anti-BiP (BD Biosciences, San Jose, CA),
and anti-PERK and anti-JNK (Cell Signaling Technologies, Danvers, MA). Secondary antibodies were
obtained from LI-COR (IRDye 680 and 800 nm, for use with the LI-COR Odyssey system [Lincoln, NE])
and Santa Cruz Biotechnology (horseradish peroxidase conjugated).

Dersh D., Jones S.M., Eletto D., Christianson J.C, & Argon Y. (2014). OS-9 facilitates turnover of nonnative GRP94 marked by hyperglycosylation. Molecular Biology of the Cell, 25(15), 2220-2234.

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Glycosylation Monitoring in Eukaryotic Cells

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The following reagents were used: kifunensine and 4-methylumbelliferyl-6-sulfo-N-acetyl-β-d-glucosaminide (Calbiochem, Billerica, MA); bafilomycin A1, MG-132, dimethyl sulfoxide (DMSO), biotin, streptavidin, brefeldin A, proline, glutamic acid, TMAO, and trehalose (Sigma, St. Louis, MO); Z-VAD(OMe)-FMK (Abcam, Cambridge, England). For Western blot analyses, the following antibodies were used: anti-FLAG M1 (Sigma); anti–14-3-3 and anti-SEL1L (Santa Cruz, Dallas, TX); anti–OS-9, anti-PDIA6, anti-calreticulin, anti-HEXA (α), anti-HEXB (β), and anti-PDIA1 (Abcam); anti-GRP94 9G10 (Enzo, Farmingdale, NY); anti-BiP (BD Biosciences, San Jose, CA); anti-GRP170 (kind gift from Linda Hendershot, St. Jude Children’s Research Hospital, Memphis, TN); anti-HA (Cell Signaling Technologies, Danvers, MA); and anti-V5 (Invitrogen, Carlsbad, CA). Secondary antibodies were obtained from Li-Cor, Lincoln, NE (IRDye 680 and 800 nm, for use with the Li-Cor Odyssey system).

Dersh D., Iwamoto Y, & Argon Y. (2016). Tay–Sachs disease mutations in HEXA target the α chain of hexosaminidase A to endoplasmic reticulum–associated degradation. Molecular Biology of the Cell, 27(24), 3813-3827.

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Immunofluorescence Analysis of Autophagy Markers

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BMDMs were fixed and stained as described in the Supplemental Experimental Proceudres using the following primary antibodies anti-LC3 (Sigma L7543); anti-LAMP1 (Santa Cruz SC-8098); anti-polyubiquitin (Enzo FK1), humanized anti-K63 and anti-K48 (Genentech), anti-proteasome 20S β2i subunit (Enzo BML-PW8150), anti-Flag M1 (Sigma), anti-p62 (Abnova H00008878-M01), anti-NBR1 (Abcam ab55474). Immunofluorescence imaging was performed using a Zeiss AxioImager M2 microscope equipped with a Photometrics CoolSnap HQ2 camera and deconvolution microscopy using AutoDeBlur (Bitplane). Imaris (7.4.0 version; Bitplane) was used for image analysis.

Franco L.H., Nair V.R., Scharn C.R., Xavier R.J., Torrealba J.R., Shiloh M.U, & Levine B. (2016). The ubiquitin-ligase Smurf1 functions in selective autophagy of M. tuberculosis and anti-tuberculous host defense. Cell host & microbe, 21(1), 59-72.

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Immunoprecipitation and Western Blotting Assay

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Immunoprecipitation and western blotting were performed as described previously [9 (link), 15 (link)]. Membranes were probed with antibodies against BCAR4 (C78-I97, see above), EGFR (clone 2.1E1, Zytomed Systems, Berlin, Germany), ERBB2 (clone 4B5, Ventana, Tucson, AZ, U.S.A.), and β-actin (clone AC15, Acris, Hiddenhausen, Germany) and Anti-Flag M1 (Sigma, Zwijndrecht, the Netherlands).

van Agthoven T., Dorssers L.C., Lehmann U., Kreipe H., Looijenga L.H, & Christgen M. (2015). Breast Cancer Anti-Estrogen Resistance 4 (BCAR4) Drives Proliferation of IPH-926 lobular Carcinoma Cells. PLoS ONE, 10(8), e0136845.

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Immunoblotting Technique for Protein Detection

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After separation by SDS-PAGE on standard polyacrylamide Tris-glycine gels, the proteins were transferred onto PVDF membranes (pore size 0.45 μm, Sigma). The membranes were blocked with 5% (w/v) dried skimmed milk in TBS (25 mM Tris-HCl pH 7.5, 150 mM NaCl) and incubated with primary antibodies followed by IRDye fluorescently labelled secondary antibodies (LI-COR). The membranes were scanned with an Odyssey near-infrared imager (LI-COR). Primary antibodies and antisera against human IRE1α LD (Shemorry et al, 2019 (link)), mouse IRE1α serum (NY200) (Bertolotti et al, 2000 (link)), hamster BiP (chicken anti-BiP (Avezov et al, 2013 (link))), eIF2α (mouse anti-eIF2α (Scorsone et al, 1987 (link))) and monoclonal anti-FLAG-M1 (Sigma) were used.
Coomassie-staining was carried out with Instant Blue (Expedeon). Signal quantitation from SDS-PAGE gels or from immunoblots was carried out using the ImageJ software (NIH). For quantitative immunoblotting (Fig. EV5B), a precast gel NuPAGE™ 4 to 12%, Bis-Tris, 1.0–1.5 mm, Mini Protein Gel (Thermo Fisher) was used.

Neidhardt L., Cloots E., Friemel N., Weiss C.A., Harding H.P., McLaughlin S.H., Janssens S, & Ron D. (2023). The IRE1β-mediated unfolded protein response is repressed by the chaperone AGR2 in mucin producing cells. The EMBO Journal, 43(5), 719-753.

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Monoclonal and Polyclonal Antibodies for β2AR

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Mouse monoclonal antibodies against β₂AR at serine 261/262 (clone 2G3 and 2E1) and at serine 355/356 (clone 10A5) were kindly provided by Dr. Richard Clark (UT Huston). Polyclonal antibodies against β₂AR (m20 and sc-570) and phosphorylated β₂AR at serine 355/356 (sc-22191R and sc-16719R) were purchased from Santa Cruz Biotechnology (SCBT, CA). Polyclonal antibodies against α₁1.2 residues 754–901 for total α₁1.2 (FP1), α₁1.2 residues 1923–1935 for phosphorylated serine 1928 site (LGRRApSFHLECLK, pS1928) and α₁1.2 residues 1694–1709 for phosphorylated serine 1700 site (EIRRAIpSGDLTAEEEL, pS1700) were made in house^{26 (link)}. Other antibodies used in the experiments include: anti-FLAG-M1 and biotinylated anti-FLAG-M2 (F3040 and F9291, Sigma, MO), biotinylated goat anti-mouse IgG, and goat anti-rabbit IgG (111–065–144 and 115–055–166, Jackson ImmunoResearch, PA).
Isoproterenol (ISO) (Sigma, MO) was freshly prepared in water for every experiment. When necessary, ICI-118551 (Sigma, MO) was prepared in water as a stock solution and added into solution to a final concentration of 1 μM.

Shen A., Nieves-Cintron M., Deng Y., Shi Q., Chowdhury D., Qi J., Hell J.W., Navedo M.F, & Xiang Y.K. (2018). Functionally distinct and selectively phosphorylated GPCR subpopulations co-exist in a single cell. Nature Communications, 9, 1050.

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