Anti dykddddk

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-DYKDDDDK is a lab equipment product that can be used to detect and purify proteins tagged with the DYKDDDDK peptide sequence, also known as the FLAG® tag. It is a highly specific and sensitive antibody that recognizes the FLAG® tag and can be used in various applications, such as Western blotting, immunoprecipitation, and immunoaffinity purification.

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Lab products found in correlation

Anti myc, by Cell Signaling Technology (3 mentions) Rnase a t1 mix, by Thermo Fisher Scientific (2 mentions) Anti flag m2 magnetic bead, by Merck Group (2 mentions) Anti cnot1, by Proteintech (2 mentions) Anti cnot6, by Cell Signaling Technology (2 mentions) Anti cnot7, by Proteintech (2 mentions) Anti tuba, by Cell Signaling Technology (2 mentions) Anti ha, by Cell Signaling Technology (2 mentions) Alexa fluor 647 secondary antibody, by Thermo Fisher Scientific (1 mentions) Fluoromount g, by Thermo Fisher Scientific (1 mentions) Anti gbp5, by Cell Signaling Technology (1 mentions) Alexa fluor 647 and alexa fluor 488 secondary antibodies, by Thermo Fisher Scientific (1 mentions) Ecl reagent, by Bio-Rad (1 mentions) Polyvinylidene fluoride (pvdf), by Bio-Rad (1 mentions) Chemidoc, by Bio-Rad (1 mentions) Image lab software, by Bio-Rad (1 mentions) Chemidoc xr, by Bio-Rad (1 mentions) Trans blot turbo transfer pack, by Bio-Rad (1 mentions) Laemmli loading buffer, by Bio-Rad (1 mentions) Bis tris precast polyacrylamide gels, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

Anti-FLAG/DYKDDDDK Tag (8 citations) Anti-DYKDDDDK Tag (6 citations) Rabbit anti-DYKDDDDK (5 citations) Anti‐FLAG (DYKDDDDK), (4 citations) Anti-DYKDDDDK Tag antibody (3 citations)

9 protocols using anti dykddddk

FH-DND1 Interactome Identification

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FH-DND1-HEK293 cells from one confluent 15-cm plate were lysed in NP40 lysis buffer (50 mM HEPES, pH 7.5, 150 mM KCl, 2 mM MgCl₂, 1 mM NaF, 0.5% (v/v) NP40, 0.5 mM DTT, complete EDTA-free protease inhibitor cocktail (Roche)) and FH-DND1 immunoprecipitated with anti-FLAG-M2 magnetic beads (SIGMA-ALDRICH, cat #M8823) in the presence or absence of 5 U/μl RNase A and 200 U/μl RNase T1 (RNase A/T1 mix, Thermo-Fisher, cat #AM2286). Magnetic beads were washed twice with NP40 lysis buffer and the immunoprecipitate eluted with SDS-gel loading buffer. Co-immunoprecipitated proteins were detected using Western Blotting with the following antibodies: anti-DYKDDDDK (Cell Signaling Technology, cat #2368), anti-CNOT1 (Proteintech, cat #14276-1-AP), anti-CNOT6 (Cell Signaling Technology, cat #13415), anti-CNOT7 (Proteintech, cat #14102-1-AP), and anti-TUBA (Cell Signaling Technology, cat #2125).

Yamaji M., Jishage M., Meyer C., Suryawanshi H., Der E., Yamaji M., Garzia A., Morozov P., Manickavel S., McFarland H.L., Roeder R.G., Hafner M, & Tuschl T. (2017). DND1 maintains germline stem cells via recruitment of the CCR4-NOT complex to target mRNAs. Nature, 543(7646), 568-572.

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FH-DND1 Interactome Identification

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Immunofluorescence Assays for Intracellular Pathogens

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For immunofluorescence assays, cells were fixed with 4% paraformaldehyde for 15 min. The cells were then permeabilized and blocked for 60 min with 0.1 to 0.2% (vol/vol) Triton X-100 in phosphate-buffered saline (PBS) (pH 7.4), typically with 3% (wt/vol) goat serum. For T. gondii intracellular growth and survival assays, cells were incubated with mouse polyclonal anti-SAG1 (DG52) (67 (link)) at 1:10,000 overnight, followed by incubation with Alexa Fluor 647 secondary antibodies (Molecular Probes) for 1 h. For GBP5 localization assays, cells were incubated with anti-SAG1 (DG52) at 1:10,000 and anti-GBP5 (Cell Signaling Technologies, no. 67798) at 1:500 overnight followed by incubation with Alexa Fluor 647 and Alexa Fluor 488 secondary antibodies (Molecular Probes) for 1 h. For ROP16 localization assays, cells were incubated with anti-SAG1 (DG52) at 1:10,000 and anti-DYKDDDDK (Cell Signaling Technologies, no. 14793) at 1:500 overnight followed by incubation with Alexa Fluor 647 and Alexa Fluor 488 secondary antibodies (Molecular Probes) for 1 h. Coverslips were mounted on slides using Fluoromount G (ThermoFisher, 00-4958-02).

Kochanowsky J.A., Thomas K.K, & Koshy A.A. (2021). ROP16-Mediated Activation of STAT6 Suppresses Host Cell Reactive Oxygen Species Production, Facilitating Type III Toxoplasma gondii Growth and Survival. mBio, 12(2), e03305-20.

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Western Blot Immunodetection of Proteins

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Proteins were transferred to nitrocellulose membrane in a wet transfer cell (50 V, 2.5 h). Membrane was blocked in 5% milk for 1 h followed by primary incubation overnight at 4 °C (1:2500 anti-myc (Cell Signaling) or 1:2500 anti-DYKDDDDK (Cell Signaling)). Membranes were washed with TBST (3x, 10 min) and incubated with secondary anti-rabbit antibody (Licor) for 1 h at room temperature.

Zaro B.W., Vinogradova E.V., Lazar D.C., Blewett M.M., Suciu R.M., Takaya J., Studer S., de la Torre J.C., Casanova J.L., Cravatt B.F, & Teijaro J.R. (2019). Dimethyl fumarate disrupts human innate immune signaling by targeting the IRAK4-MYD88 complex. Journal of immunology (Baltimore, Md. : 1950), 202(9), 2737-2746.

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Western Blot Analysis of HIF-1α Transmembrane Domains

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Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), ntHIF-1α-TMD and HIF-1α-TMD were electrophoresed and blotted onto polyvinylidene fluoride (PVDF, Bio-Rad, Hercules, CA, USA). The membrane was blocked with a blocking buffer composed of 4% bovine serum albumin in Tris-buffered saline containing Tween 20 (TBST). The membrane was then incubated overnight at 4°C with anti-DYKDDDDK (Cell Signaling, Danvers, MA, USA). After TBST wash, TBST containing anti-mouse IgG (Abcam, Cambridge, MA, USA) was added to the membrane, and the membrane was incubated for 1 h at room temperature (RT). ECL reagent (Bio-Rad) was added onto the membrane, and the chemiluminescence signal from the membrane was detected using ChemiDoc (Bio-Rad).

Shin J.S., Kim I., Moon J.S., Ho C.C., Choi M.S., Ghosh S, & Lee S.K. (2021). Intranuclear Delivery of HIF-1α-TMD Alleviates EAE via Functional Conversion of TH17 Cells. Frontiers in Immunology, 12, 741938.

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Western Blot Analysis of Protein Expression

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On the day of the analysis, cell lysates were thawed on ice and then combined with 2x Laemmli loading Buffer (BioRad) with 0.05% BME. The samples were heat denatured for 10 min at 95 °C and loaded into a Bolt™ 4 to 12% (vol/vol), Bis-Tris precast polyacrylamide gels (Invitrogen by Thermo Fisher). Gels were run at 120V in 1x MOPS Buffer and were then transferred to a 0.2 μm PVDF nitrocellulose membrane (Trans-Blot Turbo Transfer Pack, Bio-Rad) using the Trans-Blot Turbo Transfer System (13A, 25V, 7 min) (BioRad). The membrane was blocked in 5% BSA for 1 h followed by overnight incubation at 4 °C with primary antibodies (1:1000 anti-mCherry [Cell Signaling Technology] or 1:1000 anti-DYKDDDDK [Cell Signaling Technology]). Membranes were washed with TBST (3x, 5 min) and incubated with secondary anti-rabbit HRP antibody for 1 h at room temperature followed by a subsequent washing with TBST (3x, 5 min). The membrane was developed with ECL western blotting detection reagent kit (Thermo Scientific) and imaged on ChemiDoc XRS+ with Image Lab Software (Bio-Rad).

Vinogradova E.V., Zhang X., Remillard D., Lazar D.C., Suciu R.M., Wang Y., Bianco G., Yamashita Y., Crowley V.M., Schafroth M.A., Yokoyama M., Konrad D.B., Lum K.M., Simon G.M., Kemper E.K., Lazear M.R., Yin S., Blewett M.M., Dix M.M., Nguyen N., Shokhirev M.N., Chin E.N., Lairson L.L., Melillo B., Schreiber S.L., Forli S., Teijaro J.R, & Cravatt B.F. (2020). An activity-guided map of electrophile-cysteine interactions in primary human T cells. Cell, 182(4), 1009-1026.e29.

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Molecular Tools for CFTR and ER Stress Research

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Thapsigargin (Sigma‐Aldrich #T9033), 1‐deoxynojirimycin hydrochloride (Sigma‐Aldrich #D9305), kifunensine (Sigma‐Aldrich #K1140), swainsonine (Sigma‐Aldrich #S8195), Dynasore hydrate (Sigma‐Aldrich #D7693), bafilomycin A1 (Sigma‐Aldrich #B1793), MG132 (Selleckchem #S2619), and Ponceau S (Sigma‐Aldrich #P3504) were purchased commercially. CSTMP was custom‐synthesized by Cayman Chemical (Ann Arbor, MI, USA) (CAS registry number 1000672‐89‐8).
The following antibodies were acquired commercially: anti‐CFTR clone M3A7 (Millipore #05‐583, Billerica, MA, USA), anti‐HA (Cell Signaling Technology #2367, Danvers, MA, USA), anti‐Myc (Cell Signaling Technology #2276), anti‐Aldolase A (Abcam #ab78339, Cambridge, UK), anti‐Pendrin (Santa Cruz, #sc‐23779), anti‐IRE1α (Cell Signaling Technology #3294), anti‐phospho S724 IRE1α (Abcam #ab48187), anti‐TMED3 (Abcam #ab223175), anti‐TMED9 (Proteintech #21620‐1‐AP), anti‐TMED10 (Proteintech #15199‐1‐AP), anti‐TMED2 (Proteintech #11981‐1‐AP), anti‐DYK (GenScript #A00187), anti‐LC3 (Sigma #L8918), anti‐GFP (Santa Cruz #sc‐9996), anti‐DYKDDDDK (Cell Signaling Technology #2368), anti‐HA (Novus #NB600‐362), anti‐Furin (Invitrogen #PA1‐062), anti‐GM130 (BD Biosciences #610 899), anti‐Calnexin (Abcam #ab219644), anti‐GRASP55 (Abcam #ab74579) anti‐SARS‐CoV‐2 S1 (GeneTex #GTX135356), and anti‐SARS‐CoV‐2 S2 (GeneTex # GTX632604).

Park H., Seo S.K., Sim J., Hwang S.J., Kim Y.J., Shin D.H., Jang D.G., Noh S.H., Park P., Ko S.H., Shin M.H., Choi J.Y., Ito Y., Kang C., Lee J.M, & Lee M.G. (2022). TMED3 Complex Mediates ER Stress‐Associated Secretion of CFTR, Pendrin, and SARS‐CoV‐2 Spike. Advanced Science, 9(24), 2105320.

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Antibody Sources for Protein Analysis

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Anti-HA, anti-DYKDDDDK, anti-Myc, and anti-His antibodies were provided by Cell Signaling Technology (CST, Danvers, MA, USA), while Santa Cruz Biotechnology (Dallas, TX, USA) was the source of the anti-USP48 antibody. The anti-USP48, anti-HMGA2, anti-Tubulin and anti-V5 antibodies were supplied by Proteintech (Wuhan), and the PTM BioLab (Hanzhou, China) provided the anti-Ub antibody. Anti-c-Myc, anti-His, and Anti-HA agarose beads were supplied by Medchem Express (MCE) based in NJ, USA. 2-D08, MG132 and CQ were provided by SelleckChem located in Houston, TX, USA.

Cheng C., Yao H., Li H., Liu J., Liu Z., Wu Y., Zhu L., Hu H., Fang Z, & Wu L. (2024). Blockade of the deubiquitinating enzyme USP48 degrades oncogenic HMGA2 and inhibits colorectal cancer invasion and metastasis. Acta Pharmaceutica Sinica. B, 14(4), 1624-1643.

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Characterization of OTUB1 Regulation

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Full‐length OTUB1 expression constructs were purchased from ORIGENE. Point mutations to generate OTUB1 C91S were obtained by QuickChange site‐directed mutagenesis PCR (Stratagene, La Jolla). Lentiviral pLA‐CMV‐N‐Flag or pLA‐CMV‐N‐HA vectors were used to generate Flag/HA‐tagged constructs. The pMT107–6×His–ubiquitin plasmid was a generous gift from Dr. Bohmann (University of Rochester, USA). pLKO.1‐puro shGFP and pLKO.1‐puro vectors containing shRNAs targeting OTUB1 (pLKO.1‐shOTUB1_1 (TRCN0000004211), pLKO.1‐shOTUB1_2 (TRCN0000004213), pLKO.1‐shOTUB1_3 (TRCN0000004215)) were purchased from Sigma‐Aldrich.
The antibodies used: mouse monoclonal anti‐FLAG (Sigma‐Aldrich, M2), anti‐RAS (Millipore, Clone 10), anti‐vinculin (Sigma‐Aldrich, clone hVIN‐1), anti‐GAPDH (Sigma‐Aldrich, GAPDH‐71.1), anti‐p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling, 3A7, #9107), anti‐AKT (Cell Signaling, 40D4); rat monoclonal anti‐HA (Roche, 3F10); rabbit monoclonal anti‐phospho‐p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (IHC) (Cell Signaling, D13.14.4E, #4370), anti‐Ki67 (Thermo Scientific #RM‐9106‐S, clone SP6); rabbit polyclonal anti‐DYKDDDDK (Cell Signaling), anti‐OTUB1 (Bethyl Laboratories), anti‐OTUB1 (IHC) (Sigma‐Aldrich, HPA039176), anti‐phospho‐p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (Cell Signaling, #9101), anti‐pAKT (Cell Signaling, D9E), anti‐RABEX5 (Sigma‐Aldrich).

Baietti M.F., Simicek M., Abbasi Asbagh L., Radaelli E., Lievens S., Crowther J., Steklov M., Aushev V.N., Martínez García D., Tavernier J, & Sablina A.A. (2016). OTUB1 triggers lung cancer development by inhibiting RAS monoubiquitination. EMBO Molecular Medicine, 8(3), 288-303.

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