μmacs ha isolation kit

Manufactured by Miltenyi Biotec

Sourced in Germany

The μMACS HA Isolation kit is a laboratory equipment designed for the isolation and purification of proteins tagged with the hemagglutinin (HA) epitope. The kit utilizes magnetic beads coated with anti-HA antibodies to capture the HA-tagged proteins from complex samples, enabling their efficient separation and recovery.

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Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Lysis buffer, by Miltenyi Biotec (1 mentions) Anti flag m2 magnetic bead, by Merck Group (1 mentions)

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Isolation kits (10 citations)

4 protocols using μmacs ha isolation kit

Metabolic labeling of selenoproteins

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The Flp-In™ stable cell lines HA-GPx1, HA-GPx1Cys and HA-GPx1CysΔSECIS were generated as described in (8 (link)). A custom siRNA library consisting of pools of four different dual strand modified siRNAs per target (ON-TARGET plus SMART pools Custom Library, Dharmacon) or individual siSMN1 (Dharmacon) were transfected with Lipofectamine 2000 (Invitrogen) following the manufacturer's conditions. siRNAs used for qRT-PCR are listed in Supplementary Table S1. The efficiency of siRNA inhibition was tested by qRT-PCR and western blot analysis. Forty-eight hours after transfection the expression of HA-GPx1, HA-GPx1Cys and HA-GPx1CysΔSECIS was induced using DMEM/10% FCS containing 0.5 μg/ml Doxycycline and 3 nM of Na₂SeO₃. After 3 h of induction, cells were washed with Met-free medium (DMEM Glutamax Gibco). The medium was replaced with Met-free DMEM containing 100 μCi/ml [³⁵S]-methionine for 1 h. The radioactive medium was removed and cells were lysed immediately, HA-tagged proteins were purified using a μMACS HA isolation kit (Miltenyi Biotec) and analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography.

Gribling-Burrer A.S., Leichter M., Wurth L., Huttin A., Schlotter F., Troffer-Charlier N., Cura V., Barkats M., Cavarelli J., Massenet S, & Allmang C. (2017). SECIS-binding protein 2 interacts with the SMN complex and the methylosome for selenoprotein mRNP assembly and translation. Nucleic Acids Research, 45(9), 5399-5413.

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Co-immunoprecipitation of zDHHC17 interactions

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For co-immunoprecipitation assays, HEK293T cells in 24-well plates were co-transfected with murine zDHHC17 in HA-pEF-BOS plasmid (or empty vector) and EGFP, EGFP-PAI-RBP1, or EGFP-SLAIN1 plasmids in triplicate wells. Following 24 h, cells in each well were lysed in 100 μl of lysis buffer (Miltenyi Biotech) and lysates from three identical wells were pooled for co-immunoprecipitation assays. Protein isolation was performed using μMACS HA Isolation kit (Miltenyi Biotec), according to the manufacturer's protocol; however, to preserve binding of proteins to HA-zDHHC17, all washes were performed in lysis buffer.

Lemonidis K., MacLeod R., Baillie G.S, & Chamberlain L.H. (2017). Peptide array-based screening reveals a large number of proteins interacting with the ankyrin-repeat domain of the zDHHC17 S-acyltransferase. The Journal of Biological Chemistry, 292(42), 17190-17202.

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Cloning and Co-Immunoprecipitation of hnRNP L and MBNL1

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The long (NCBI Ref. Seq: XM_024451480.1) and short (NCBI Ref. Seq: NM_001005335.2) hnRNP L open reading frames (ORFs) were cloned into the pIRES-hrGFP-1a plasmid (Stratagene; San Diego, CA) via standard PCR-based cloning into Not1 and Xho1 restriction sites. Human MBNL1 ORF sequence (NCBI Ref. Seq: NM_021038.5) was subcloned into the pIRES-hrGFP-2a plasmid (Stratagene) into the Not1 and Xho1 restriction sites using PCR-based cloning. Plasmids were transfected into HEK293T (GenHunter Corp.; Nashville, TN) at 1 μg each in 10 cm plates at approximately 50% confluency using Lipofectamine 2000 (Invitrogen; Carlsbad, CA) following the manufacturer’s protocol. Co-immunoprecipitations (co-IPs) were performed using magnetic beads conjugated either to HA (μMACS HA Isolation Kit; Miltenyi Biotech; Bergisch Gladbach, Germany; Cat# 130-091-122) or the FLAG epitope (anti-FLAG M2 Magnetic Beads; Millipore-Sigma; Burlington, MA; Cat#M8823-1ML) following the manufacturer’s protocol. Approximately 200 μg of total protein lysate was used for immunoprecipitation reactions. Western immunoblots were performed as previously described^{32 (link)}.

Alexander M.S., Hightower R.M., Reid A.L., Bennett A.H., Iyer L., Slonim D.K., Saha M., Kawahara G., Kunkel L.M., Kopin A.S., Gupta V.A., Kang P.B, & Draper I. (2021). hnRNP L is essential for myogenic differentiation and modulates myotonic dystrophy pathologies. Muscle & nerve.

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Cohesin Binding Assay on pUC19

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Cohesin loading assays were done as described in (14 (link)) using the pUC19 plasmid. Topologically bound DNA-cohesin complexes were immunoprecipitated using a μMACS HA Isolation kit (Miltenyi Biotec). Following incubation with Pst I and/or protein digestion, the recovered DNA was analyzed by electrophoresis on a 0.8% (w/v) TAE agarose gel in 1× TAE and visualized as described above.

Gutierrez-Escribano P., Newton M.D., Llauró A., Huber J., Tanasie L., Davy J., Aly I., Aramayo R., Montoya A., Kramer H., Stigler J., Rueda D.S, & Aragon L. (2019). A conserved ATP- and Scc2/4-dependent activity for cohesin in tethering DNA molecules. Science Advances, 5(11), eaay6804.

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