Anti ha

Manufactured by Santa Cruz Biotechnology

Sourced in United States, United Kingdom, Germany

Anti-HA is a laboratory reagent used for the detection and purification of proteins tagged with the hemagglutinin (HA) epitope. It provides a reliable and specific way to identify and isolate HA-tagged proteins in various experimental applications.

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Close spelling variants of this product

Anti-HA antibody (91 citations) Anti-HA (sc-7392, (21 citations) Anti-HA beads (5 citations) Polyclonal anti-HA antibody (SC-805 (4 citations) Anti-hemagglutinin (HA) (4 citations) Mouse anti-HA 12CA5 (3 citations) Anti-HA 12CA5 (3 citations) Mouse monoclonal anti-HA antibody (3 citations) Mouse anti‐HA (sc‐7,392, (3 citations) Anti-HA rabbit polyclonal antibody (3 citations)

247 protocols using anti ha

Immunochemistry Antibody Characterization

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Sources of commercial antibodies were as follows: anti-FUS (Santa Cruz, Dallas, TX, USA; 4H11), anti-Pur-alpha (Abcam, Cambridge, UK; ab77734), anti-Pur-alpha (Abcam; ab79936), anti-HA (Santa Cruz; Y-11), anti-HA (Santa Cruz; F-7), anti-Flag (Sigma-Aldrich, St. Louis, MO, USA; M2 and M2 affinity gel), anti-Flag fluorescein isothiocyanate (FITC) conjugated (Sigma-Aldrich; M2), anti-Phospho-eIF2-alpha (Cell Signaling Technology, Beverly, MA, USA; D9G8), anti-eIF2-alpha (Cell Signaling Technology; D7D3), anti-cleaved caspase-3 (Cell Signaling Technology; Asp175), anti-NeuN (Merck Millipore, Billerica, MA, USA; A60), anti-TIAR (BD Biosciences, Erembodegem, Belgium), anti-GAPDH (Chemicon-Merck Millipore), anti-puromycin 3RH11 monoclonal antibody (Kerafast, Boston, MA, USA), anti-FUS (Abcam; ab84078), anti-Islet-1/2 (DSHB, Iowa City, IA, USA; 39.4D5). Mouse monoclonal antibody specific for ribosomal protein S19 were prepared in our laboratory.^{31 (link)} FITC, Rhodamine, and aminomethylcoumarin-conjugated affinity-purified secondary anti'bodies, selected for absent cross-reaction, were from Jackson ImmunoResearch (West Grove, PA, USA).

Di Salvio M., Piccinni V., Gerbino V., Mantoni F., Camerini S., Lenzi J., Rosa A., Chellini L., Loreni F., Carrì M.T., Bozzoni I., Cozzolino M, & Cestra G. (2015). Pur-alpha functionally interacts with FUS carrying ALS-associated mutations. Cell Death & Disease, 6(10), e1943-.

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Western Blotting and Immunoprecipitation Assay Protocol

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Cells were lysed using RIPA buffer (Tris-HCl pH 7.6 50 mM, deoxycholic acid sodium salt 0.5%, NaCl 140 mM, NP40 1%, EDTA 5 mM, NaF 100 mM, sodium pyrophosphate 2 mM and protease inhibitors). Lysates were separated on 8% acrylamide gel and immunoblotted using standard procedures. The following antibodies were used: anti-Arrb1 K-16 (sc-8182; Santa Cruz Biotechnology), anti-Nanog (Cosmo Bio Co, Japan), anti-Actin I-19 (sc-1616; Santa Cruz Biotechnology), anti-β-III-Tubulin (MAB 1637 Millipore), anti-Gli1 H-300 (sc-20,687; Santa Cruz Biotechnology), anti-acetyl-Gli1 (Lys518) (Eurogentec) [32 (link)], anti-p300 C-20 (sc-585; Santa Cruz Biotechnology), anti-FLAG M2-Peroxidase (HRP) (A8592 Sigma), anti-HA (sc-7392 Santa Cruz), anti-Gli2 H-300 (sc-28,674; Santa Cruz Biotechnology), anti-Smo N-19 (sc-6366; Santa Cruz Biotechnology), anti-Sox2 (MAB4343 Millipore). HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) were used in combination with enhanced chemo-luminescence (ECL Amersham).
For immunoprecipitation assay antibody sources and concentrations used were: Protein G Plus-Agarose (sc-2002; Santa Cruz Biotechnology); anti-FLAG M2 Affinity Gel (Sigma A2220, IP 30⌠l), anti-FLAG M2-Peroxidase (HRP) (A8592 Sigma, western blotting 1:5000), anti-HA (sc-7392 Santa Cruz, 1:1000); anti-myc-HRP.

Miele E., Po A., Begalli F., Antonucci L., Mastronuzzi A., Marras C.E., Carai A., Cucchi D., Abballe L., Besharat Z.M., Catanzaro G., Infante P., Di Marcotullio L., Canettieri G., De Smaele E., Screpanti I., Locatelli F, & Ferretti E. (2017). β-arrestin1-mediated acetylation of Gli1 regulates Hedgehog/Gli signaling and modulates self-renewal of SHH medulloblastoma cancer stem cells. BMC Cancer, 17, 488.

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Immunoprecipitation and Co-immunoprecipitation Assay

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Cells were lysed with lysis buffer containing 50 mM Tris-HCl (pH 7.8), 150 mM NaCl, 1% NP-40, 0.1% deoxycholate and protease inhibitor cocktails (Roche). For DNase and RNase treatment, cell lysates were treated with 40 U ml⁻¹ DNase and 10 μg ml⁻¹ RNase at 37 °C for 30 min. Lysates were immunoprecipitated using anti-Flag (Sigma-Aldrich, F2426) or anti-HA (Santa Cruz, sc-805 AC) antibodies. For co-immunoprecipitation of endogenous proteins, E13.5 mouse brains were homogenized in the above lysis buffer. Lysates were immunoprecipitated using TLX antibody, followed by immunoblotting using indicated antibodies. To determine whether Dpi disrupts the TLX-Drosha interaction, constructs expressing Dpi (TLX residues 341–359) or a control peptide (TLX residues 201–223), together with HA-TLX and Flag-Drosha were transfected into HEK293T cells. Cell lysates were immunoprecipitated with Flag antibody (Sigma-Aldrich, F2426), followed by immunoblotting with anti-HA (1:500, Santa Cruz, sc-805) or anti-Flag antibody (1:500, Sigma-Aldrich, F1804). Images in Figs 1a,b, 2c,d,f and 5b–e have been cropped for presentation. Full size images are presented in Supplementary Figs 8–10.

Murai K., Sun G., Ye P., Tian E., Yang S., Cui Q., Sun G., Trinh D., Sun O., Hong T., Wen Z., Kalkum M., Riggs A.D., Song H., Ming G.L, & Shi Y. (2016). The TLX-miR-219 cascade regulates neural stem cell proliferation in neurodevelopment and schizophrenia iPSC model. Nature Communications, 7, 10965.

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BES1-UBP12 Protein Interaction Assay

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Seven-day-old seedlings of 35S:BES1-myc/UBQ10:UBP12-HA were incubated with 50 μM MG132 (Calbiochem, cat. no. 474790) in the presence or absence of 1 μM eBL (Sigma, cat. no. E1641) for 6 h at 22°C under light conditions. Treated plants were collected and homogenized with coIP buffer consisting of 50 mM Tris–HCl (pH 7.4), 100 mM NaCl, 0.2% Triton X-100, 1 mM DTT, and protease inhibitor cocktail (Roche, cat. no. 04693159001). An aliquot of the total protein was saved as an input sample. Total protein extracts were incubated with protein A-agarose for 1 h at 4°C to remove adventitious binding proteins. The antibodies anti-myc (Proteintech, cat. no. 60003-2-Ig), anti-HA (Santa Cruz, cat. no. SC-805), and anti-GFP (Santa Cruz, cat. no. SC-9996) were added to the treated protein extracts for 2 h at 4°C. The antibody/protein complex, which was retrieved by incubating with 20 μl of protein A-agarose for 2 h at 4°C, was washed with a coIP buffer 5 times. The co-immunoprecipitated proteins were separated by SDS–PAGE followed by immunoblotting assays with anti-HA (Santa Cruz, cat. no. SC-805), anti-myc (Proteintech, cat. no. 16286-1-AP), and anti-Actin (ProteinTech, cat. no. 66009-1-Ig) antibodies using the ECL Prime Western Blotting System (GE Healthcare, cat. no. RPN2232). All assays were repeated in three independent experiments.

Park S.H., Jeong J.S., Zhou Y., Binte Mustafa N.F, & Chua N.H. (2022). Deubiquitination of BES1 by UBP12/UBP13 promotes brassinosteroid signaling and plant growth. Plant Communications, 3(5), 100348.

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Protein Complex Identification via Co-IP

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Total cell lysate was collected with IP-lysis buffer (150 mM NaCl, 20 mM NaCl, 20 mM HEPES, pH 7.2, 10 mM NaF, 1 mM EDTA, 1% NP-40, 1 mM Na₃V0₄, 1 mM PMSF, 1 DTT, and proteinase inhibitor cocktail) at 72 h, and subjected to immunoblotting with anti-myc (1:3000, Santa Cruz, CA, USA) and anti-HA (1:1000, Santa Cruz, CA, USA) monoclonal antibody. Co-immunoprecipitation was performed with 25 μl Protein A/G PLUS-Agarose (Santa Cruz, CA, USA) and anti-myc monoclonal antibody (1 μg) to pull-down the complex, which was then immunoblotted with anti-HA monoclonal antibody.

Lien H.W., Yuan R.Y., Chou C.M., Chen Y.C., Hung C.C., Hu C.H., Hwang S.P., Hwang P.P., Shen C.N., Chen C.L., Cheng C.H, & Huang C.J. (2016). Zebrafish cyclin Dx is required for development of motor neuron progenitors, and its expression is regulated by hypoxia-inducible factor 2α. Scientific Reports, 6, 28297.

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Immunoprecipitation and Western Blotting of Transfected or Infected Cells

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Transfected 293 cells or viral infected brain slices were homogenized in 0.5% NP-40 lysis buffer (0.5% NP-40, 10% glycerol, 50 mM Tris, pH 7.6, 150 mM NaCl, 50mM NaF, 0.1mM EDTA, and 0.1mM Na₃VO₄, 1mM phenylmethylsulfonyl fluoride, and protease inhibitor tablet), then lysates were ultra-centrifuged (200,000 × g) at 4°C for 60 min. Supernatant fractions were incubated with anti-HA (Santa Cruz Biotechnology, sc-805) or anti-NR1 (NeuroMab clone N308/48) for overnight at 4°C, followed by incubation with 50 μl of protein A/G plus agarose (Santa Cruz Biotechnology) for 1 hr at 4°C. Immunoprecipitates were washed three times with lysis buffer containing 0.2 M NaCl, then boiled in 2× SDS loading buffer for 5 min, and separated on 7.5% SDS-polyacrylamide gels. Western blotting experiments were performed with anti-Flag (1:1000, Sigma, F3165), anti-PSD-95 (1:1000, NeuroMab, 75-028), anti-NR1 (1:500, NeuroMab clone N308/48) or anti-HA (1:200, Santa Cruz Biotechnology, sc-805).

Qin L., Liu W., Ma K., Wei J., Zhong P., Cho K, & Yan Z. (2016). The ADHD-linked Human Dopamine D4 Receptor Variant D4.7 Induces Over-Suppression of NMDA Receptor Function in Prefrontal Cortex. Neurobiology of disease, 95, 194-203.

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Immunological and Biochemical Profiling

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CD4-Percp (eBioscience, 46-0041), IL-4-APC (MACS, 130-103-002), IL-5-PE (eBioscience, 12-7052), and IL-13-PE Cy7 (eBioscience, 25-7133) were used for flow cytometry. Anti-IL-33 (Abcam, Ab54385) was used for immunofluorescence of mice lung tissues. Anti-PTRF (CST, 46379), anti-Flag (Sigma-Aldrich, M2), anti-HA (Santa Cruz, sc-7392), anti-GFP (Santa Cruz, sc-9996), anti-pTyr (Santa Cruz, sc-7020), anti-β-Actin (Sungene biotech, KM9001T), anti-α-Tubulin (Sungen biotech, KM9003T), and anti-Lamin B (CST, 13435) were used for immunoblotting.

Ni Y., Hao J., Hou X., Du W., Yu Y., Chen T., Wei Z., Li Y., Zhu F., Wang S., Liang R., Li D., Lu Y., Liao K., Li B, & Shi G. (2018). Dephosphorylated Polymerase I and Transcript Release Factor Prevents Allergic Asthma Exacerbations by Limiting IL-33 Release. Frontiers in Immunology, 9, 1422.

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Immunofluorescence Imaging of Protein Localization in Cells

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HeLa cells were seeded on coverslips and transfected with plasmids. After 48 h, cells were fixed with 4% formaldehyde, and incubated with anti-HA (Santa Cruz Biotechnology) or anti-Myc (Santa Cruz Biotechnology) antibody, followed by secondary goat anti-rabbit antibody coupled to Alexa488 (Invitrogen) and goat anti-mouse antibody coupled to Alexa546 (Invitrogen). The nucleus was stained with DAPI (Invitrogen). Coverslips were mounted and fluorescence was visualized with 40× magnification on a confocal laser scanning microscope (Carl Zeiss, Inc.), and pictures were analyzed with the ZEN 2009 software.
To count cells with mitotic defects, HeLa cells grown on coverslips were transfected with the indicated siRNA and plasmids for subsequent rescue. After fixation and permeabilization, cells were stained with monoclonal anti-β-tubulin-Cy3™ (Sigma) for 2 h, and the nucleus was counterstained with DAPI. Cells with mitotic defects were counted at 40× magnification with a fluorescence microscope (Nikon eclipse TE 2000-U).

Das T., Park J.K., Park J., Kim E., Rape M., Kim E.E, & Song E.J. (2017). USP15 regulates dynamic protein–protein interactions of the spliceosome through deubiquitination of PRP31. Nucleic Acids Research, 45(8), 4866-4880.

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Eukaryotic and Prokaryotic Expression of Atx3 and Related Proteins

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For eukaryotic expression, Atx3-I, Atx3-IC and their mutants were cloned into a FLAG-pcDNA3.0 vector, while P97, Ub and NEDD8 were cloned into HA pcDNA3.0. For prokaryotic expression, the C-terminal fragments of Atx3-II (22Q, 6Q) were cloned into pET-22b(+), while Atx3_46Q-IIC was cloned into pGBTNH, which encodes the fusion protein with a GB1 domain in the N terminus57 (link). P97-ND1 (residues 1–458) and P97N (1–213) were cloned into pET-22b(+), while Ub was cloned into pET-3a. The anti-FLAG antibody was from Sigma; anti-HA, anti-Ub and anti-actin were from Santa Cruz; and anti-GAPDH was from Zen BioScience. All the secondary antibodies were purchased from Jackson Immuno-Research. PVDF membranes were obtained from PerkinElmer Life Sciences, and ECL detection kit for proteins was from Thermoscientific.

Yang H., Li J.J., Liu S., Zhao J., Jiang Y.J., Song A.X, & Hu H.Y. (2014). Aggregation of polyglutamine-expanded ataxin-3 sequesters its specific interacting partners into inclusions: Implication in a loss-of-function pathology. Scientific Reports, 4, 6410.

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Quantifying Epigenetic Modifications in Cells

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pCMV-HA-JMJD3 plasmid was obtained from Addgene (#24167). Lentiviral shRNAs (Extended Data Table 5) were provided by the Vector Core at the University of Michigan or kindly provided by Dr. Arul Chinnaiyan (University of Michigan). Antibodies including monoclonal anti-EZH2 (1:2000, BD Biosciences, 612667), anti-H3K27me3 (1:1000, Millipore, 07-449), H3K9me2 (1:2000, ab1220, Abcam), H3K9me3 (1:500, ab8898, Abcam), H3K4me1 (ab8895, Abcam), H3K4me2 (ab194678, Abcam), H3K4me3 (ab1012, Abcam), anti-Histone H3 (1:2000, Cell Signaling, 9715), anti-HA (1:200, Santa Cruz Biotechnology, sc-805), anti-DNMT1 (1:250, Abcam, ab13537) were used for Western blotting. Anti-human CXCR3 (1C6) blocking antibody was prepared from mouse hybridoma (ATCC, #HB-12330) by Hybridoma Core at the University of Michigan.

Peng D., Kryczek I., Nagarsheth N., Zhao L., Wei S., Wang W., Sun Y., Zhao E., Vatan L., Szeliga W., Kotarski J., Tarkowski R., Dou Y., Cho K., Hensley-Alford S., Munkarah A., Liu R, & Zou W. (2015). Epigenetic silencing of Th1 type chemokines shapes tumor immunity and immunotherapy. Nature, 527(7577), 249-253.

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