Ezview red anti ha

Manufactured by Merck Group

EZview Red Anti-HA is a laboratory equipment product designed for the detection and purification of proteins that are tagged with the hemagglutinin (HA) epitope. The product provides a simple and efficient method for immunoaffinity capture and visualization of HA-tagged proteins.

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Lab products found in correlation

Ecl plus kit, by Cytiva (2 mentions) Anti flag affinity gel, by Merck Group (2 mentions) Protein a g bead, by Santa Cruz Biotechnology (2 mentions) Anti flag m2 affinity agarose beads, by Merck Group (1 mentions) Ezview anti flag m2 agarose, by Merck Group (1 mentions) Complete protease inhibitors and phosstop, by Roche (1 mentions) Fugene 6 transfection reagent, by Promega (1 mentions) Protein a g agarose bead, by Santa Cruz Biotechnology (1 mentions) Immobilion p membrane, by Merck Group (1 mentions) Immobilon p membrane, by Merck Group (1 mentions)

Close spelling variants of this product

Anti-HA (475 citations) EZview Red Anti-HA Affinity Gel (83 citations) EZview Red anti-HA or anti-FLAG affinity gel (5 citations) EZview Red Anti-HA Affinity Gel beads (5 citations) EZview Red anti-HA agarose affinity gel (4 citations) EZviewTM Red Anti‐HA beads (4 citations) EZview™ Red anti-HA affinity beads (3 citations) EZview Red Anti-HA Affinity matrix (2 citations)

6 protocols using ezview red anti ha

PCDH19 Interacts with CDH Proteins

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PCDH19 (pCMV-cMyc-PCDH19 or pCMV-FLAG-PCDH19) interaction with PCDH1
(pEF1a-PCDH1-cMyc-CMV-EGFP), PCDH9 (pEF1a-PCDH9-HA-CMV-EGFP) or CDH13
(pEF1a-CDH13-FLAG-CMV-EGFP) was tested by immunoprecipitation (IP) in HEK293T
cells transfected with the various combinations of plasmids expressing the
epitope-tagged proteins. HEK293T cells were plated at 5 × 10⁵/well in
six-well culture plates. The next day, cells were transfected with the
expression plasmids using Lipofectamine 3000 reagent, harvested 24 h
post-transfection, lysed in cold PCDH19 lysis buffer (50 mM Tris–HCl pH 7.5, 150
mM NaCl, 0.2% Triton X-100, 2 mM EDTA, 0.01% SDS, 50 mM NaF, 0.1 mM
Na₃VO₄ and 1× Protease inhibitor/No EDTA (Merck),
sonicated (1 × 30% amplitude, 10 s on Sonic’s Vibra-Cell VCX) and centrifuged 15
000 r.p.m. at 4°C for 15 min. Lysis buffer with 0.4% Triton X-100 was used for
the PCDH19–CDH13 IPs. The 10% lysate was saved as input and the remainder was
subjected to IP using either EZview red anti-HA or anti-FLAG M2 agarose affinity
beads (Sigma-Aldrich). IP complexes were eluted using 1× SDS protein-loading
buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol and 5% β-mercaptoethanol [50 (link)]) at 95°C for 5 min and stored at −80°C until western blot
analysis.

Mincheva-Tasheva S., Pfitzner C., Kumar R., Kurtsdotter I., Scherer M., Ritchie T., Muhr J., Gecz J, & Thomas P.Q. (2024). Mapping combinatorial expression of non-clustered protocadherins in the developing brain identifies novel PCDH19-mediated cell adhesion properties. Open Biology, 14(4), 230383.

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Immunoprecipitation of Protein Complexes

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Sub confluent HEK293 cells were transiently transfected with 3μg of plasmids using Fugene 6 transfection reagent (Promega, Madison, WI)(25 ). Seventy-two hours after transfection, cells were lysed in IP-lysis buffer (Pierce) supplemented with protease inhibitors cocktail (cOmplete, Protease and PhosSTOP inhibitors; Roche). Cell extracts (500μg) were immunoprecipitated with EZview anti-Flag M2 Agarose (Sigma) or EZview Red Anti-HA (Sigma) Affinity Gels overnight at 4°C and processed for immunoblot analysis. For endogenous co-IP experiments, MEC1 or Mino cells were processed as above, and complexes were captured by using Protein A/G-Agarose beads (Santa Cruz, Dallas, TX) at room temperature for 1–2 hours. The beads were washed three times with lysis buffer and immunoblotted.

Tantawy S.I., Sarkar A., Hubner S., Tan Z., Wierda W.G., Eldeib A., Zhang S., Kornblau S, & Gandhi V. (2023). Mechanisms of MCL-1 protein stability induced by MCL-1 antagonists in B-cell malignancies. Clinical cancer research : an official journal of the American Association for Cancer Research, 29(2), 446-457.

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Western Blot Immunoprecipitation Analysis

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Cell lysates were separated on sodium dodecylsulfate (SDS)-polyacrylamide (7–15%) gels, and transferred on to Immobilion-P membranes (Millipore; Bedford, MA). Membranes were blocked with Tris-buffered saline solution containing 5% skim milk and 0.1% Tween-20 for 1 hour and incubated with a primary antibody overnight at 4 °C. The membranes were incubated with a horseradish peroxidase-conjugated secondary antibody for 1 hour and visualized using the ECL Plus kit (Amersham Biosciences; Piscataway, NJ). To analyze protein interactions, cell lysates were incubated with anti-FGFR2 antibodies overnight at 4 °C, and the immune complexes were precipitated using protein A/G beads (Santa Cruz, CA). Otherwise, cell lysates were incubated with EZview Red anti-HA or anti-FLAG affinity gel (Sigma-Aldrich) overnight at 4 °C. Precipitated immunocomplexes were eluded with a denaturing 2xSDS sample buffer and subjected to immunoblotting. Immunoblots were quantified using the ImageJ program (NIH, Bethesda, Maryland).

Lee J.E., Shin S.H., Shin H.W., Chun Y.S, & Park J.W. (2019). Nuclear FGFR2 negatively regulates hypoxia-induced cell invasion in prostate cancer by interacting with HIF-1 and HIF-2. Scientific Reports, 9, 3480.

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Ypt7Δ Cells Pexophagy Induction

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To induce peroxisome proliferation, ypt7Δ cells expressing Pex5-HA from its endogenous promoter were pre-cultured in YPD medium overnight and then shifted to oleate medium for around 16 h. Subsequently, cells were transferred to SD-N medium to induce pexophagy. 250 OD₆₀₀ of cells was harvested and resuspended in 4 mL of Zymolyase solution (Nacalai, 07663–91) after cells were starved for 1 h. The following co-immunoprecipitation was performed as described previously [44]. Sigma-Aldrich EZview Red Anti-HA (Sigma, E6779) was used to pull down Pex5-HA. The eluate was separated by SDS-PAGE and used for mass spectroscopy analysis at the UC San Diego core facility.

Wang X., Wang P., Zhang Z., Farré J.C., Li X., Wang R., Xia Z., Subramani S, & Ma C. (2019). The autophagic degradation of cytosolic pools of peroxisomal proteins by a new selective pathway. Autophagy, 16(1), 154-166.

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Immunoprecipitation of HA-Pex19 Complex

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Cells were grown in YPD medium overnight to approximately 2 OD₆₀₀/mL and then switched to methanol medium. After 6 h of induction, 250 OD₆₀₀ of cells was harvested and resuspended in 4 mL Zymolyase solution. The cells were spheroplasted at 30 °C for 30 min. Next, they were spun down and resuspended in 1.5 mL lysis buffer [20 mM Hepes/KOH (pH 7.4), 0.15 M NaCl, 5 mM NaF, 20 mM EDTA (pH 8.0), 50 µg leupeptin/mL, 50 µg aprotinin/mL, 1 mM PMSF, PIC yeast]. The cells were lysed by vortexing in the presence of acid-washed glass beads. The lysate was centrifuged at 20,000g for 20 min at 4 °C to remove the cytosolic fraction. The pellet was resuspended in 1.5 mL lysis buffer (with 1% Chaps) and placed on a rotator at 4 °C for solubilization. The samples were then spun at 20,000g for 20 min. The supernatant was collected and mixed with specified antibody affinity matrix (Sigma-Aldrich EZview Red Anti-HA) to pull down HA-Pex19. They were left to incubate overnight before washing the beads four times with lysis buffer. One hundred twenty microliters of sample buffer was added to the beads and placed in boiling water for 5 min. The eluate was analyzed via SDS-PAGE and Western blotting for anti-HA, anti-Pex3, anti-Pex2, and anti-Pex17 antibodies.

Farré J.C., Carolino K., Stasyk O.V., Stasyk O.G., Hodzic Z., Agrawal G., Till A., Proietto M., Cregg J., Sibirny A.A, & Subramani S. (2017). A New Yeast Peroxin, Pex36, a Functional Homolog of Mammalian PEX16, Functions in the ER-to-Peroxisome Traffic of Peroxisomal Membrane Proteins. Journal of molecular biology, 429(23), 3743-3762.

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Western Blot and Co-Immunoprecipitation Analysis

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Proteins were separated on SDS-polyacrylamide gels and transferred to Immobilon-P membranes (Millipore; Bedford, MA). The membranes were blocked with 5% skim milk, incubated overnight at 4 °C with a primary antibody, and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h, and visualized using the ECL-plus kit (Amersham Biosciences; Piscataway, NJ). To analyze protein interactions, cell lysates were incubated with anti-FIH, anti-NAA10, or IgG overnight at 4 °C, and the immune-complexes were pulled down with protein A/G beads (Santa Cruz Biotechnology). Otherwise, cell lysates were incubated with EZview Red anti-HA or anti-FLAG affinity gel (Sigma-Aldrich) at 4 °C for 4 h. The bound proteins were eluted in a denaturing SDS sample buffer or HA/FLAG-tag peptides, and loaded on SDS-PAGE. All Western blotting experiments were performed three or more times.

Kang J., Chun Y.S., Huh J, & Park J.W. (2018). FIH permits NAA10 to catalyze the oxygen-dependent lysyl-acetylation of HIF-1α. Redox Biology, 19, 364-374.

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