Ha y 11

Manufactured by Santa Cruz Biotechnology

Sourced in United States, Canada

HA (Y-11) is an antibody specific for the hemagglutinin (HA) protein of the influenza virus. It can be used for the detection and analysis of the HA protein in research applications.

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Lab products found in correlation

Horseradish peroxidase, by GE Healthcare (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Alexa fluor 594, by Thermo Fisher Scientific (2 mentions) Anti rabbit igg, by Thermo Fisher Scientific (2 mentions) Anti mouse igg, by Thermo Fisher Scientific (2 mentions) Cul1 2h4c9, by Merck Group (2 mentions) Anti mouse igg or protein a, by GE Healthcare (2 mentions) Anti rabbit igg, by GE Healthcare (2 mentions) Flag tag (flag), by Cell Signaling Technology (2 mentions) Flag m2, by Fortrea (2 mentions) β actin, by Fortrea (2 mentions) Myc 9e10, by Santa Cruz Biotechnology (2 mentions) Abl 8e9, by BD (2 mentions) Ptyr p tyr 100, by Cell Signaling Technology (2 mentions) Crkii c 18, by BD (2 mentions) P3xflag cmv24, by Merck Group (1 mentions) D3n8f, by Cell Signaling Technology (1 mentions) Flag m2, by Merck Group (1 mentions) β actin 1 19, by Santa Cruz Biotechnology (1 mentions) Gapdh, by Merck Group (1 mentions)

13 protocols using ha y 11

Myc and Fbw7 Mutational Analysis

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Myc and all mutants thereof are in pCS2+ vector containing a single HA tag at the N terminus. Fbw7 and all mutants thereof are in p3xFLAG-CMV24 (Sigma-Aldrich), except HA-Fbw7, which was cloned into pCS2+ with a single N-terminal HA tag. All mutations were generated by the QuikChange method (Stratagene) and verified by sequencing. Fbw7 ∆F has the entire F-box deleted, while Fbw7 ∆D has a deletion of five critical amino acids in the dimerization domain (14 (link)). The following antibodies were used: FLAG: M2 (Sigma-Aldrich), HA: Y11 (Santa Cruz Biotechnology), Myc: D3N8F (Cell Signaling Technology), pT58: EPR17923 (Abcam ab185655), pS62: ERP17924 (Abcam ab185656), pT244: in-house mouse monoclonal, Fbw7: A301-720A and A301-721A (Bethyl Laboratories), proliferating cell nuclear antigen (PCNA): PC-10 (Santa Cruz Biotechnology), and GSK3: 368662 (Calbiochem).

Welcker M., Wang B., Rusnac D.V., Hussaini Y., Swanger J., Zheng N, & Clurman B.E. (2022). Two diphosphorylated degrons control c-Myc degradation by the Fbw7 tumor suppressor. Science Advances, 8(4), eabl7872.

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Monitoring ISG15 Deconjugation by USP18

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USP18-deficient mouse embryonic fibroblasts were stimulated with 250 U/ml IFNβ for 24h to induce ISGylation. To generate lysates with HA-ISG15- or HA-ct-ISG15-conjugated substrates, HEK 293T cells were transfected with vectors encoding hUbe1L, hUbcH8 and mHerc6 together with the plasmid encoding HA-ISG15 or HA-ct-ISG15. The cells were lysed in 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Triton-X100. 20 µg of the lysate was incubated with 20 µg of USP18 or the USP18 variants. The reaction was performed in 50 mM Na₂HPO₄, 500 mM NaCl, 10 mM DTT pH 7.9 for 0 and 2 h at 37°C. The samples were analyzed by Western blot with antibodies directed against ISG1562 (link), HA (Y-11, Santa Cruz, Santa Cruz, USA), β-Actin (I-19, Santa Cruz, Santa Cruz, USA) and GAPDH (Millipore, Billerica, USA).

Basters A., Geurink P.P., Röcker A., Witting K.F., Tadayon R., Hess S., Semrau M.S., Storici P., Ovaa H., Knobeloch K.P, & Fritz G. (2017). Structural basis of the specificity of USP18 toward ISG15. Nature Structural & Molecular Biology, 24(3), 270-278.

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Antibodies for Western Blotting and Immunofluorescence

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Example 3

Mouse monoclonal antibodies were purchased from Invitrogen (CUL1-2H4C9: #32-2400), Sigma (FLAG-M2: #F3165, β-actin: #A-1978), Covance (HA-16612: #MMS-101P) and Santa Cruz (CD147 8D6: #sc-21746): Rabbit polyclonal antibodies were from Millipore (MCT1: #AB-3538P), Bethyl (DDB1: #A300-462A, CUL4A: #A300-739A), Sigma (FLAG, #F7425), Cell Signaling (α/β-tubulin: #2148, IKZF1: #5443, IKZF3: #12720), Proteintech (CUL4B: #12916-1-AP) and Santa Cruz (HA Y-11: #sc-805. A polyclonal antibody against CRBN was generated by immunizing rabbits with a mixture of two peptides containing amino acids 1-19 and 424-437 of human CRBN (MAGEDQQDAAHNMGNHLPC (SEQ. ID NO: 1) and CPTIDPTEDEISPDK (SEQ ID NO: 2), respectively). Secondary antibodies (anti-rabbit IgG, anti-mouse IgG or protein-A coupled with horseradish peroxidase) were from GE Healthcare. Secondary antibodies (anti-rabbit IgG and anti-mouse IgG) coupled to Alexa Fluor 594 or Alexa Fluor 488 for immunofluorescence and flow cytometry were from Life technologies.

US10987345B2. Method for assessing the efficacy of IMiDs and composition or combination for use in treating IMiD sensitive diseases (2021-04-27). Klinikum Rechts Der Isar Der Technischen Universitat Munchen [DE]. Inventors: Florian Bassermann [DE], Ruth Eichner [DE], Vanesa Fernandez-Saiz [DE].

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Multimodal Protein Analysis Protocol

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β‐catenin (610154, BD), caspase‐3 (9661L, Cell Signalling), Cyclin D1 (2978S, Cell Signalling), DVL2 (10B5) (sc‐8026, Santa Cruz), GAPDH (sc‐47724, Santa Cruz), Flag (F3165, SIGMA), HA (Y‐11) (sc‐7392, Santa Cruz), LGR4 (C‐12) (sc‐390630, Santa Cruz), Lysozyme (A0099, Dako), c‐Myc (9E10) (sc‐40, Santa Cruz), NEDD4 (sc‐25508, Santa Cruz), NEDD4L (4013S, Cell Signalling), SNAP (P9310S, NEB), Sox9 (AB5335, Millipore) and V5 (ab27671) were used in immunohistochemistry, immunoprecipitations or Western blot analysis.

Novellasdemunt L., Kucharska A., Jamieson C., Prange‐Barczynska M., Baulies A., Antas P., van der Vaart J., Gehart H., Maurice M.M, & Li V.S. (2019). NEDD4 and NEDD4L regulate Wnt signalling and intestinal stem cell priming by degrading LGR5 receptor. The EMBO Journal, 39(3), e102771.

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Immunofluorescence Staining of Cellular Proteins

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Antibodies against Myc (9E10, 1:200), HA (Y-11, 1:100), TRI (V-22, 1:200) were obtained from Santa Cruz Biotechnology. Antibody against clathrin (1:200) was from BD bioscience. Antibodies against Smad3 (1:200) and p-Smad3 (1:200) were from Cell Signaling Technology. Mouse and rabbit Alexa Fluor 488-, 555- and 647- conjugated secondary antibodies (1:500), Aleax Fluor 488- and 568-conjugated transferrin (20 μg/mL) and Aleax Fluor 555-conjugated Cholera Toxin B (CTB-555 20 μg/mL) were all from Molecular Probes. SB431542 (20 μM) was from Sigma. Dynasore (80–160 μM) was from Selleckchem. Human TGFβ1 (10 ng/mL) was from R&D.

Li N., Yang Y., He K., Zhang F., Zhao L., Zhou W., Yuan J., Liang W, & Fang X. (2016). Single-Molecule Imaging Reveals the Activation Dynamics of Intracellular Protein Smad3 on Cell Membrane. Scientific Reports, 6, 33469.

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Heterologous expression of PKD proteins

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Human PKD2L1 cDNA (accession #: NM_016112) was inserted into vector pCHGF^{61 (link)} for efficient expression in Xenopus laevis oocyte. Flag tag was then added before the N-terminus of the PKD2L1 for immunodetection. HA-tagged human PKD2 (NM_000297) plasmid was constructed as we previously reported^{5 (link)}. Mutagenesis was carried out using QuikChange Lightning Site-Directed Mutagenesis kit (Agilent Technologies, La Jolla, CA) and confirmed by sequencing. Sequences of primers used in this study are included in Supplementary Table 2. Rabbit FLAG (D-8, 1:2000), HA (Y-11, 1:2000) and mouse β-actin (C-4, 1:4000) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA) for immunoblotting. Secondary antibodies against rabbit (NA934-1ML, 1:2000) or mouse (NA931-1ML, 1:2000) lgG were from GE Healthcare (Waukesha, WI).

Zheng W., Yang X., Hu R., Cai R., Hofmann L., Wang Z., Hu Q., Liu X., Bulkley D., Yu Y., Tang J., Flockerzi V., Cao Y., Cao E, & Chen X.Z. (2018). Hydrophobic pore gates regulate ion permeation in polycystic kidney disease 2 and 2L1 channels. Nature Communications, 9, 2302.

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Yeast Protein Extraction and Detection

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Protein extracts of S. cerevisiae were prepared either by glass bead lysis or alkaline lysis followed by TCA precipitation. Proteins were separated by SDS-PAGE, transferred to PVDF or nitrocellulose membranes and detected using antibodies according to standard protocols. Commercially available antibodies were used to detect individual proteins: myc (9E10, Santa Cruz Biotechnology), HA (Y-11, Santa Cruz Biotechnology), PAP (Sigma-Aldrich), Clb2 (y-180, Santa Cruz Biotechnology), Pgk1 (Invitrogen).

Peplowska K., Wallek A.U, & Storchova Z. (2014). Sgo1 Regulates Both Condensin and Ipl1/Aurora B to Promote Chromosome Biorientation. PLoS Genetics, 10(6), e1004411.

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Chromatin Immunoprecipitation of HA-BRCA1

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Fragmented chromatin was immunoprecipitated using a chromatin immunoprecipitation assay kit (EMD Millipore). 293T cells transfected with pBabe or pBabe HA BRCA1 were cross-linked by formaldehyde. Fragmented chromatin was immunoprecipitated with rabbit polyclonal HA (Y-11; Santa Cruz Biotechnology) or rabbit immunoglobulin G (IgG) (Santa Cruz Biotechnology). The quantitative real-time PCR was performed using SYBR Select Master Mix (Applied Biosystems). The primer sequences for the ChIP assay were described previously (21 (link)).

Chiyoda T., Hart P.C., Eckert M.A., McGregor S.M., Lastra R.R., Hamamoto R., Nakamura Y., Yamada S.D., Olopade O.I., Lengyel E, & Romero I.L. (2017). Loss of BRCA1 in the cells of origin of ovarian cancer induces glycolysis: A window of opportunity for ovarian cancer chemoprevention. Cancer prevention research (Philadelphia, Pa.), 10(4), 255-266.

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Immunoblotting of Replication Factors

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ORC2 antibody was from Marcel Méchali (IGH, Montpellier). Pol η, Mcm2 (AbCam), PCNA and β-actin (Sigma), HA (Y-11, Santa Cruz Biotechnology), Myc9B11, PR-Set/Set8, P-p53 (Ser15), P-H2AX (Ser139; Cell Signalling) and Cdt1 (Millipore).

Tsanov N., Kermi C., Coulombe P., Van der Laan S., Hodroj D, & Maiorano D. (2014). PIP degron proteins, substrates of CRL4Cdt2, and not PIP boxes, interfere with DNA polymerase η and κ focus formation on UV damage. Nucleic Acids Research, 42(6), 3692-3706.

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Quantifying Receptor Tyrosine Kinase Signaling

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pTyr (P-Tyr-100) was from Cell Signaling; Dok1 (A-3), pDok1 (Tyr362), Actin (I-19) and HA (Y-11) were from Santa Cruz Biotechnology; CrkII (C-18) and Abl (8E9) were from BD Biosciences; RasGAP (B4F8) was from Upstate Biotechnology.

Ng K.Y., Yin T., Machida K., Wu Y.I, & Mayer B.J. (2014). Phosphorylation of Dok1 by Abl family kinases inhibits CrkI transforming activity. Oncogene, 34(20), 2650-2659.

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