Anti ha immunoprecipitation kit

Manufactured by Merck Group

Sourced in United States

The Anti-HA Immunoprecipitation Kit is a laboratory tool used to isolate and purify proteins tagged with the hemagglutinin (HA) epitope. The kit contains antibodies and reagents necessary for the immunoprecipitation process, which allows researchers to selectively capture and concentrate HA-tagged proteins from complex samples for further analysis.

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Lab products found in correlation

Flag immunoprecipitation kit, by Merck Group (2 mentions) Silver staining kit, by Beyotime (2 mentions) Co ip lysis buffer, by Beyotime (2 mentions) Cellytic m, by Merck Group (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Cellytic m, by Thermo Fisher Scientific (1 mentions) Halt phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions) Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Anti flag anti haantibodiesand protein a g agarose beads, by Santa Cruz Biotechnology (1 mentions) Flag immunoprecipitation kit flagipt1, by Merck Group (1 mentions) Protein a g agarose bead, by Santa Cruz Biotechnology (1 mentions)

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Anti-HA (475 citations) Immunoprecipitation Kit (14 citations)

6 protocols using anti ha immunoprecipitation kit

Purification of Viral Proteins

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HEK293 were transfected with the appropriate expression vectors using liposomal reagent Transfast^TM following manufacturer’s instructions, washed and lysed 48 h later with CelLytic M (Sigma-Aldrich, USA). Lysates were cleared by centrifugation.
For purification of HA-tagged pUL56, the cell-free reaction was performed with Anti-HA Immunoprecipitation Kit according to the manufacturer’s protocol (Sigma-Aldrich, USA).
For purification of His-tagged pUL89, the cell-free reaction was performed with Ni resin (Clontech, USA).
All proteins were concentrated approximately 5-fold using Pall centrifugal filters (Pall, USA), and protein concentration was determined by the Bradford method using bovine serum albumin (Sigma-Aldrich, USA) as standard protein.

Ligat G., Jacquet C., Chou S., Couvreux A., Alain S, & Hantz S. (2017). Identification of a short sequence in the HCMV terminase pUL56 essential for interaction with pUL89 subunit. Scientific Reports, 7, 8796.

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Anti-HA Immunoprecipitation of iPSC Cardiomyocytes

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Immunoprecipitation (IP) assays were performed with an anti-HA immunoprecipitation kit (IP0010, Sigma-Aldrich). Briefly, transfected iPSC cardiomyocytes cultured in 24-well plates were lysed in CelLytic M reagent supplemented with protease inhibitor cocktail (Thermo Fisher Scientific) and Halt phosphatase inhibitor (Thermo Fisher Scientific). Cell lysates were incubated overnight at 4 °C with anti-HA-affinity gel in a mini-spin column. After wash, retained proteins were eluted from columns by incubation at 95 °C for 10 min with NuPAGE LDS sample buffer followed by centrifugation. The resulting IP samples were analyzed by Western blot and dot-blot as described above.

Cejas R.B., Tamaño-Blanco M., Fontecha J.E, & Blanco J.G. (2022). Impact of DYRK1A Expression on TNNT2 Splicing and Daunorubicin Toxicity in Human iPSC-Derived Cardiomyocytes. Cardiovascular Toxicology, 22(8), 701-712.

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ESCO2-hnRNPA1 Protein Interaction

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FLAG-tagged ESCO2, HA-tagged hnRNPA1, and their mutant proteins were purified from HEK293T cells using a FLAG Immunoprecipitation Kit (FLAGIPT1, Sigma) or Anti-HA Immunoprecipitation Kit (IP0010, Sigma). Recombinant ESCO2 proteins were incubated with recombinant hnRNPA1 or its mutants in 30 μL reaction buffer (50 mMNaCl, 50 mMTris-HCl [pH 8.0], 4 mM MgCl₂, 1 mM DTT, 0.1 mM EDTA, 10% glycerol) at 37 °C for 30 min.

Zhu H.E., Li T., Shi S., Chen D.X., Chen W, & Chen H. (2021). ESCO2 promotes lung adenocarcinoma progression by regulating hnRNPA1 acetylation. Journal of Experimental & Clinical Cancer Research : CR, 40, 64.

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Immunoprecipitation of pVHL and Ubiquitin Complexes

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For pVHL or ubiquitin immunoprecitations, 25 μl of goat-Anti-Rabbit IgG (New England Biolabs, S1432S) or goat-Anti-Mouse IgG (New England Biolabs, S1431S) magnetic beads were pre-incubated (2 hour at RT) with 5 μg of anti-VHL antibody (Thermo Fisher Scientific PA5–27322) or an anti-total-ubiquitin antibody (Abcam ab128424). Cells were lysed using an IP lysis buffer (Thermo Fisher Scientific 87787) and added to the magnetic beads overnight at 4C. The magnetic beads were then washed five times with a Tris-EDTA buffer to remove non-bound proteins and the bound proteins were separated from the beads by adding SDS buffer and boiling the protein for 5 min. Immunoprecipitated samples were subjected to standard SDS-PAGE and Western blot. For HA-tag immunoprecitations, an Anti-HA immunoprecipitation kit (Sigma-Aldrich IP0010) was used per the manufacturer’s instruction. Immunoprecipitated samples were subjected to SDS-PAGE and Western blot as described above.

Esmerats J.F., Villa-Roel N., Kumar S., Gu L., Salim M.T., Ohh M., Taylor W.R., Nerem R.M., Yoganathan A.P, & Jo H. (2019). Disturbed flow increases UBE2C via loss of miR-483-3p, inducing aortic valve calcification by the HIF1α pathway in endothelial cells. Arteriosclerosis, thrombosis, and vascular biology, 39(3), 467-481.

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Identifying ESCO2-interacting Proteins via Co-IP and MS

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HEK293T cells were transfected with Flag-ESCO2 vector for 48h using Lipofectamine 2000. Treated HEK293T cells were lysed in Co-IP lysis buffer (P0013, Beyotime, Shanghai, China). Co-IP was performed using anti-FLAG/anti-HAantibodiesand protein A/G agarose beads(sc-2003, Santa Cruz)to extractthe complexes. Gel bands were detected using a silver staining kit (P0017S, Beyotime) combined with MS following the manufacturer's protocol. According to a previously published method (27) , the peptides of the bands were analyzed using nano-LC-MS/MS. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identi er PXD023527 and PXD23600.
In vitro acetylation assay FLAG-tagged ESCO2, HA-tagged hnRNPA1, and their mutant proteins were puri ed from HEK293T cells using a FLAG Immunoprecipitation Kit (FLAGIPT1, Sigma) or Anti-HA Immunoprecipitation Kit (IP0010, Sigma). Recombinant ESCO2 proteins were incubated with recombinant hnRNPA1 or its mutants in 30 μL reaction buffer (50 mMNaCl, 50 mMTris-HCl [pH 8.0], 4 mM MgCl 2 , 1 mM DTT, 0.1mM EDTA, 10% glycerol) at 37°C for 30 min.

Zhu H., Li T., Shi S., Chen D., Chen W., & Chen H. (2021). ESCO2 promotes lung adenocarcinoma progression by regulating hnRNPA1 acetylation.

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ESCO2 Interacts with hnRNPA1 In Vitro

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HEK293T cells were transfected with Flag-ESCO2 vector for 48h using Lipofectamine 2000. Treated HEK293T cells were lysed in Co-IP lysis buffer (P0013, Beyotime, Shanghai, China). Co-IP was performed using anti-FLAG/anti-HA antibodies and protein A/G agarose beads (sc-2003, Santa Cruz) to extract the complexes. Gel bands were detected using a silver staining kit (P0017S, Beyotime) combined with MS following the manufacturer's protocol. According to a previously published method [26], the peptides of the bands were analyzed using nano-LC-MS/MS.
In vitro acetylation assay FLAG-tagged ESCO2, HA-tagged hnRNPA1, and their mutant proteins were puri ed from HEK293T cells using a FLAG Immunoprecipitation Kit (FLAGIPT1, Sigma) or Anti-HA Immunoprecipitation Kit (IP0010, Sigma). Recombinant ESCO2 proteins were incubated with recombinant hnRNPA1 or its mutants in 30 μL reaction buffer (50 mM NaCl, 50 mM Tris-HCl [pH 8.0], 4 mM MgCl 2 , 1 mM DTT, 0.1 mM EDTA, 10% glycerol) at 37°C for 30 min.

Zhu H., Li T., Shi S., Chen D., Chen W., & Chen H. (2020). ESCO2 promotes lung adenocarcinoma progression by regulating hnRNPA1 acetylation.

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