Ezview red anti ha affinity beads

Manufactured by Merck Group

EZview™ Red anti-HA affinity beads are a laboratory product used for the immunoaffinity purification of HA-tagged proteins. They consist of agarose beads coated with a high-affinity anti-HA antibody. The beads can be used to capture and isolate HA-tagged proteins from complex mixtures, such as cell lysates or culture supernatants, for further analysis or downstream applications.

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Lab products found in correlation

Anti flag m2 affinity beads, by Merck Group (2 mentions) Recombinant atm, by Merck Group (1 mentions)

Close spelling variants of this product

EZview Red Anti-HA Affinity Gel beads EZviewTM Red Anti‐HA beads EZview Red Anti-HA EZview anti-HA beads Anti-HA beads HA beads Anti-HA

3 protocols using ezview red anti ha affinity beads

Protein Immunoprecipitation and GST Pull-Down Assay

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Cells were lysed with NETN buffer containing protease inhibitors on ice for 30 min, then sonicated for 15 s. Following centrifugation, the clarified lysates were incubated with protein G or protein A agarose beads coupled with antibody against the indicated proteins for 8 h at 4°C. Beads were washed with NETN buffer three times and analyzed by western blot. For tagged protein IP, cell lysates were incubated with Anti-Flag M2 Affinity beads (Sigma) for 3 h at 4°C, or EZview™ Red anti-HA affinity beads (Sigma) for 8 h at 4°C. Precipitates were then washed and immunoblotted with the indicated antibodies. For the BRCA1 or CtIP GST-pull down assay, GST-BRCA1 fragments fusion proteins or GST-CtIP-N were expressed in Escherichia coli. Purified fusion proteins were immobilized on glutathione Sepharose 4B beads and incubated with cell lysates at 4°C. The samples were separated by SDS-PAGE and analyzed by western blot.

Chen Y., Liu H., Zhang H., Sun C., Hu Z., Tian Q., Peng C., Jiang P., Hua H., Li X, & Pei H. (2016). And-1 coordinates with CtIP for efficient homologous recombination and DNA damage checkpoint maintenance. Nucleic Acids Research, 45(5), 2516-2530.

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Kinase Assays for Chk1, TLK1, and ATM

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In vitro Chk1 or TLK1 kinase assay was performed in kinase buffer D (20 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 25 µM ATP, 8 µCi of [γ³²P] ATP). 200 ng of recombinant Chk1 (#7735; Biovision) or immunoprecipitated HA-TLK1 was incubated with 1 µg recombinant His-ASF1A for 30 min at 30ºC and the reaction stopped by addition of Laemmli buffer. UCN-01 was directly added to the reaction tube at 100 nM concentration to inhibit Chk1. Reactions were separated on 13% SDS-polyacrylamide gels. To obtain HA-TLK1 protein, HA-TLK1 expressing plasmids were transfected into HEK293T cells, and the cells were lysed in lysis buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5 mM EDTA, 0.5 mM EGTA, 5 mM MgCl2, 10 mM NaF, 0.1 mM sodium vanadate, and protease inhibitors) after 48 hr. 150 µg of lysate was mixed and incubated with EZview Red Anti-HA Affinity beads (E6779; Sigma) for 2 hr, and the beads were washed twice with lysis buffer followed by twice with kinase buffer. In vitro ATM kinase assay was performed using 200 ng of recombinant ATM (14–933; Sigma) in kinase buffer E (10 mM Tris-HCl (pH 7.5) 50 mM KCl 10 mM MgCl₂ 10 mM MnCl₂ 1 mM DTT 200 µM ATP, 4 µCi of [γ³²P] ATP).

Lee K.Y, & Dutta A. (2021). Chk1 promotes non-homologous end joining in G1 through direct phosphorylation of ASF1A. Cell reports, 34(4), 108680.

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Immunoprecipitation and GST-pulldown Assays

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Cells were lysed with NETN buffer (20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA and 0.5% NP-40) containing protease inhibitors on ice for 30 min. Following sonication, cell lysates were clarified by centrifugation and incubated with protein G or protein A agarose beads coupled with antibody against the indicated proteins for 8 h at 4 °C. Beads were then washed with NETN buffer three times and analysed by western blot. For tagged protein IP, cell lysates were incubated with Anti-Flag M2 Affinity beads (Sigma) for 3 h at 4 °C, EZview Red anti-HA affinity beads (Sigma). Precipitates were then washed and immunoblotted with the indicated antibodies. For the BRCA1 GST-pull-down assay, GST-BRCA1 fragments fusion proteins were expressed in Escherichia coli. Purified fusion proteins were immobilized on glutathione Sepharose 4B beads and incubated with cell lysates at 4 °C. The samples were separated by SDS–polyacrylamide gel electrophoresis (PAGE) and analysed by western blot. The uncropped versions of western blots are shown in Supplementary Fig. 7.

Zhang H., Liu H., Chen Y., Yang X., Wang P., Liu T., Deng M., Qin B., Correia C., Lee S., Kim J., Sparks M., Nair A.A., Evans D.L., Kalari K.R., Zhang P., Wang L., You Z., Kaufmann S.H., Lou Z, & Pei H. (2016). A cell cycle-dependent BRCA1–UHRF1 cascade regulates DNA double-strand break repair pathway choice. Nature Communications, 7, 10201.

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