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Anti ha tag antibody

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The Anti-HA-tag antibody is a laboratory tool used for the detection and identification of proteins that are tagged with the HA (hemagglutinin) epitope. The HA-tag is a widely used protein tag that allows for the specific recognition and visualization of the tagged protein in various experimental techniques.

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Lab products found in correlation

Hrp conjugation kit, by Abcam (1 mentions) Anti flag and anti myc antibodies, by Merck Group (1 mentions) Normal rabbit igg, by Merck Group (1 mentions) Pcdna3.1 myc his vector, by Thermo Fisher Scientific (1 mentions) Rapamycin hy 10219, by MedChemExpress (1 mentions) Anti myc tag antibody 2278, by Cell Signaling Technology (1 mentions) Mg132 m7449, by Merck Group (1 mentions) Dc protein assay reagent, by Bio-Rad (1 mentions) Sepharose beads, by Cell Signaling Technology (1 mentions) Prism 7, by GraphPad (1 mentions) Protease and phosphatase inhibitor cocktail, by Roche (1 mentions) β actin ac 15, by Merck Group (1 mentions) Anti rabbit alexa594, by Thermo Fisher Scientific (1 mentions) Kdm2b, by Merck Group (1 mentions) Myc tag, by Cell Signaling Technology (1 mentions) α sma, by Merck Group (1 mentions) In cell analyzer 6000, by GE Healthcare (1 mentions) Protease inhibitor tablet, by Roche (1 mentions) Dc protein assay, by Bio-Rad (1 mentions) Lsm 880 microscope, by Zeiss (1 mentions)

14 protocols using anti ha tag antibody

1

Antibody Production and Characterization for Myocilin

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Anti-TIMP3 antibodies for immunofluorescent immunohistochemistry were purchased from Novus Biologicals (Littleton, CO, USA). Anti-FLAG and anti-myc antibodies were from Sigma-Aldrich Corp. (St. Louis, MO, USA); anti-HA-tag antibody was from Cell Signaling Technology (Danvers, MA, USA). Polyclonal rabbit antiserum against mouse myocilin was described previously.11 (link) Purified anti-myocilin antibodies were purified from anti-myocilin rabbit serum through an antigen-specific affinity purification. Normal rabbit IgG was purchased from Sigma-Aldrich Corp. Affinity-purified anti-myocilin antibodies were labeled with horseradish peroxidase (HRP) using the HRP conjugation kit (Abcam, Cambridge, MA, USA) according to the manufacturer's instructions. Human myocilin, myocilin-ΔC, and myocilin-ΔN constructs tagged with the FLAG epitope were previously described.34 (link) Mouse Timp3 cDNA was cloned into the pcDNA3.1/Myc-His vector (Invitrogen, Carlsbad, CA, USA). Human full-length TIMP2, TIMP3, and TIMP4 cDNAs were cloned into the pcDNA3 vector in-frame with hemagglutinin tag (HA).
Joe M.K., Lieberman R.L., Nakaya N, & Tomarev S.I. (2017). Myocilin Regulates Metalloprotease 2 Activity Through Interaction With TIMP3. Investigative Ophthalmology & Visual Science, 58(12), 5308-5318.
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2

Monoclonal Anti-PEDV N Antibody Protocol

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The monoclonal anti-PEDV N antibody was stocked in our laboratory (48 (link)). Anti-Flag-tag antibody (F1804), chloroquine phosphate (CQ; PHR1258), 3-Methyladenine (3-MA; M9281), and MG132 (M7449) were obtained from Sigma-Aldrich. Antibodies against ACTB/β-actin (66009-1-lg), LC3 (14600-1-AP), NDP52 (12229-1-AP), MARCHF8 (14119-1-AP), FUBP3 (10623-1-AP), GST-tag (10000-0-AP), GAPDH (60004-1-lg), and TRAF3 (66310-1-lg), along with HRP-labeled anti-mouse (SA00001-1) and anti-rabbit (SA00001-2) IgG antibodies could be acquired from Proteintech Group. Bafilomycin A1 (Baf A1; 54645), anti-pIRF3 antibody (4947), anti-pTBK1 antibody (5483), anti-MYC-tag antibody (2278), and anti-HA-tag antibody (3724) were bought from Cell Signaling Technology. Human MARCHF8 siRNA (SC-90432), human NDP52 siRNA (SC-93738) as well as an anti-ubiquitin antibody (SC-8017) were acquired from Santa Cruz Biotechnology. In addition, Rapamycin (HY-10219) was bought from MedChemExpress.
Dong S., Kong N., Wang C., Li Y., Sun D., Qin W., Zhai H., Zhai X., Yang X., Ye C., Ye M., Liu C., Yu L., Zheng H., Tong W., Yu H., Zhang W., Tong G, & Shan T. (2022). FUBP3 Degrades the Porcine Epidemic Diarrhea Virus Nucleocapsid Protein and Induces the Production of Type I Interferon. Journal of Virology, 96(13), e00618-22.
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3

Immunoprecipitation and Western Blotting Analysis

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Cell lysates were prepared using NP40 lysis buffer (50 mM Tris, pH7.5; 1% Nonidet P-40; 150 mM NaCl; 10% Glycerol) containing protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). Protein concentrations were measured with DC Protein Assay Reagents (Bio-Rad, Hercules CA, USA). For immunoprecipitation, equal amounts of cell lysates were incubated with Sepharose beads linked to anti-HA-Tag antibody (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. Immunoprecipitated protein complexes were separated on SDS-PAGE gel and analyzed by Western blotting using following antibodies: anti-phosphotyrosine P-Tyr-100, anti-HA-Tag, (Cell Signaling Technology, Danvers MA, USA), anti-Ras GAP (Santa Cruz Biotechnology, Dallas TX, USA). Band intensities were quantified using ImageJ software (Schneider et al., 2012 (link)). Statistical analyses were performed using Prism 7 software (GraphPad Software, La Jolla CA, USA).
Lukasik J., Scott T.M., Andryshak D, & Farrah S.R. (2000). Influence of Salts on Virus Adsorption to Microporous Filters. Applied and Environmental Microbiology, 66(7), 2914-2920.
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4

Immunofluorescence and Immunoblotting of Protein Markers

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Cells were seeded in 96-well imaging plates and fixed the following day with 4% paraformaldehyde (PFA) for 15 minutes. Permeabilization was performed using TritonX (0.1%in PBS) for 10 min followed by incubation with blocking solution (1% BSA, 0.1% Gelatin Fish in PBS) for 30 min. Incubation with Anti-HA-tag antibody (Cell Signaling, 3724, 1:1000) or α-SMA (Sigma clone 1A4, 1:2000) was performed in blocking buffer for 2 hours at RT. Cells were washed, incubated with secondary anti-rabbit Alexa-594 (Invitrogen) and counter stained with DAPI. Image acquisition was performed using IN Cell analyzer 6000 (GE Healthcare Life Sciences). For protein lysates cells were incubated with RIPA buffer supplemented with protease inhibitors (Protease inhibitor tablets, Roche) for 30 min and cleared by centrifugation (15 min 14.000 rpms 4C). Protein was quantified using the DC protein assay (BioRad). The following antibodies were used for immunoblotting: β-ACTIN (ac-15, Sigma), KDM2B (Millipore, 09-864) HA-tag (Cell Signaling, 3724) and Myc-tag (Cell Signaling, 2276).
Banito A., Li X., Laporte A.N., Roe J.S., Sanchez-Vega F., Huang C.H., Dancsok A.R., Hatzi K., Chen C.C., Tschaharganeh D.F., Chandwani R., Tasdemir N., Jones K.B., Capecchi M.R., Vakoc C.R., Schultz N., Ladanyi M., Nielsen T.O, & Lowe S.W. (2018). The SS18-SSX oncoprotein hijacks KDM2B-PRC1.1 to drive synovial sarcoma. Cancer cell, 33(3), 527-541.e8.
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5

Immunolocalization of HA-tagged SLC26A1

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Two days after injection with cRNA (10 ng) encoding WT or mutant HA-tagged SLC26A1, oocytes were fixed with 1% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 10 minutes at 4°C and washed 3 times with cold PBS. Oocytes were blocked for 1 hour at room temperature in PBS containing 1% bovine serum albumin (PBS-BSA) and subsequently incubated for 1 hour at room temperature with anti-HA tag antibody (Cell Signaling Technology, catalog 3724) in PBS-BSA, after which the oocytes were washed 3 times with cold PBS. Antibody-labeled oocytes were then incubated for 1 hour at room temperature with secondary antibodies coupled to Alexa Fluor 555 (Molecular Probes), washed 3 times with cold PBS, and stored at 4°C until imaging. Confocal images were taken with a Zeiss LSM880 microscope using ZEN software. For the quantification of immunofluorescence microscopy images, ImageJ was used.
Pfau A., López-Cayuqueo K.I., Scherer N., Wuttke M., Wernstedt A., González Fassrainer D., Smith D.E., van de Kamp J.M., Ziegeler K., Eckardt K.U., Luft F.C., Aronson P.S., Köttgen A., Jentsch T.J, & Knauf F. (2023). SLC26A1 is a major determinant of sulfate homeostasis in humans. The Journal of Clinical Investigation, 133(3), e161849.
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6

Endothelial LRP1 Overexpression Assay

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In a subset of experiments, primary brain endothelial cells isolated from 2-mo-old Lrp1lox/lox; Tie2-Cre mice were placed in 12-well plates and incubated with Ad.mLRP1 or Ad.GFP adenovirus at concentration of 107 PFU/ml. Cells were harvested 72 h after virus transduction for LRP1 minigene Western blot analysis using anti-HA tag antibody (catalog number 372S; Cell Signaling Technology).
Nikolakopoulou A.M., Wang Y., Ma Q., Sagare A.P., Montagne A., Huuskonen M.T., Rege S.V., Kisler K., Dai Z., Körbelin J., Herz J., Zhao Z, & Zlokovic B.V. (2021). Endothelial LRP1 protects against neurodegeneration by blocking cyclophilin A. The Journal of Experimental Medicine, 218(4), e20202207.
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7

Immunohistochemical Analysis of N-cadherin, Vimentin, and HA-Tag in Tumor Tissues

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Tumor tissues were fixed in 4% paraformaldehyde and embedded in paraffin. After incubation with blocking buffer, sections were incubated with a rabbit anti-N-cadherin antibody (#GTX112733; Genetex, Irvine, CA, USA; 1:1000 dilution), goat polyclonal anti-vimentin antibody (sc-7557; Santa Cruz Biotechnology, Dallas, TX, USA; 1:50 dilution), or anti-HA-Tag antibody (#3724; Cell Signaling Technology, Inc., Danvers, MA, USA; 1:500 dilution) overnight at 4°C. After washing, sections were incubated with donkey anti-goat IgG or bovine anti-rabbit IgG, and the slides were stained with an ABC kit for color development (Santa Cruz, sc-2018). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and immunohistochemistry were carried out with assistance from the Translational Pathology Core Laboratory (TPCL) of UCLA. Images were analyzed with a Zeiss AX10 microscope.
Cheng M., Ho S., Yoo J.H., Tran D.H., Bakirtzi K., Su B., Tran D.H., Kubota Y., Ichikawa R, & Koon H.W. (2014). Cathelicidin suppresses colon cancer development by inhibition of cancer associated fibroblasts. Clinical and Experimental Gastroenterology, 8, 13-29.
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8

Generation of Mutant Ras-Expressing Cell Lines

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To generate pooled clones of NIH/3T3 cells stably expressing N-rasG12V, N-rasG12V-C51Y, K-rasG12V, and K-rasG12V-R164Q, cells were transduced with lentiviral particles for expression of HA-tagged Ras-proteins under CMV promoter and GFP-Puromycin marker under RSV promoter (AMS Biotechnology, Abingdon, UK). As a control, cells stably expressing only GFP-Puromycin marker were used. Selection with puromycin (1 μg/ml) started 72 hr after infection. After selection, cells were expanded and used in assays as indicated. Western blotting with anti-HA-tag antibody (Cell Signalling, C29F4) was performed to verify stable and comparable protein expression levels of Ras mutants.
Šolman M., Ligabue A., Blaževitš O., Jaiswal A., Zhou Y., Liang H., Lectez B., Kopra K., Guzmán C., Härmä H., Hancock J.F., Aittokallio T, & Abankwa D. (2015). Specific cancer-associated mutations in the switch III region of Ras increase tumorigenicity by nanocluster augmentation. eLife, 4, e08905.
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9

Annexin A10 Ubiquitination Regulation

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The 293T cells were co-transfected with a combination of pBabe-Cul4A-myc-his and pCMV6-ANXA10-GFP (OriGene) with or without pRK5-HA-Ubiquitin-WT (Addgene, Cambridge, MA, USA). All the cells were treated with 10 µg/mL of MG132 for 24 h prior to lysis. Anti-GFP antibody was used for immunoprecipitation. Anti-HA tag antibody (Cell Signaling, Danvers, MA, USA) was used for the western blot analysis.
Hung M.S., Chen Y.C., Lin P.Y., Li Y.C., Hsu C.C., Lung J.H., You L., Xu Z., Mao J.H., Jablons D.M, & Yang C.T. (2019). Cul4A Modulates Invasion and Metastasis of Lung Cancer through Regulation of ANXA10. Cancers, 11(5), 618.
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10

Detection and Quantification of IL-15/IL-15Rα Complex

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An immunoblotting assay was performed to detect the IL-15/IL-15Rα complex. Cell lysates (from virus-infected 293T cells) were prepared with RIPA Lysis and Extraction Buffer (ThermoFisher) containing protease/phosphatase inhibitor cocktail (Halt™). Protein concentration was assessed using a Rapid Gold BCA Protein Assay Kit (Pierce™). Anti-HA-tag antibody (Cell Signaling Technology (Cell Signaling Technology, clone: C29F4) used as a primary antibody was added for overnight incubation at 4°C, followed by incubation with anti-rabbit secondary antibody (Cell Signaling Technology) for one hour at room temperature. β-actin was used as a loading control.
An enzyme-linked immunosorbent assay (ELISA) was performed to quantify the IL-15/IL-15Rα complex secreted into the supernatants from various virus-infected GBM cell lines, using a DuoSet ELISA kit (R&D, Catalog No: DY6924) according to the manufacturer’s instructions.
Ma R., Lu T., Li Z., Teng K.Y., Mansour A.G., Yu M., Tian L., Xu B., Ma S., Zhang J., Barr T., Peng Y., Caligiuri M.A, & Yu J. (2021). An oncolytic virus expressing IL-15/IL-15Rα combined with off-the-shelf EGFR-CAR NK cells targets glioblastoma. Cancer research, 81(13), 3635-3648.
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