μmacs separator

Manufactured by Miltenyi Biotec

Sourced in Germany

The μMACS Separator is a compact, easy-to-use magnetic separation device designed for small-scale applications. It utilizes magnetic fields to separate magnetically labeled cells or particles from a sample suspension.

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7 protocols using μmacs separator

GFP Immunoprecipitation from HEK293T Cells

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For GFP-immunoprecipitation (IP), GFP MicroBeads, μ-columns, and μMACS Separator (Miltenyi Biotec) were used. μ-columns were permeabilized with 10% Triton X-100 and washed twice with IP lysis buffer (100 mM NaCl, 50 mM Tris, 5 mM EGTA, 1% CHAPS, pH 7.5, containing protease inhibitor). Transfected HEK293T cells were lysed for 20 min on ice in IP lysis buffer, sonicated for 10 min, and centrifuged for 20 min at 13,000 rpm at 4 °C. Next, 30 μl of GFP MicroBeads was added to 500 μl cell lysate and incubated for 2 h at 4 °C. After adding the cell lysates to the μ-columns, the μ-columns were washed 5 times with IP lysis buffer, and proteins were eluted from the μ-column using hot (95 °C) SDS sample buffer. For RNase treatment, 100 μg/ml RNase A (Invitrogen) was added to the IP lysis buffer. Eluates were analyzed by SDS-PAGE and subsequent Western blot.

Walsh M.B., Janzen E., Wingrove E., Hosseinibarkooie S., Muela N.R., Davidow L., Dimitriadi M., Norabuena E.M., Rubin L.L., Wirth B, & Hart A.C. (2020). Genetic modifiers ameliorate endocytic and neuromuscular defects in a model of spinal muscular atrophy. BMC Biology, 18, 127.

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GFP-tagged protein purification protocol

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Cells were lysed in lysis buffer (150 mM NaCl; 1% Triton X-100; 50 mM Tris-HCl, pH 8.0; 1 mM PMSF; Roche protease inhibitor; and a phosphatase inhibitor cocktail) on ice for 10 min. After centrifugation at 13,000 rpm for 10 min at 4° C, 1 mg of protein lysate was incubated with 50 μl of μMACS anti-GFP microbeads (Miltenyi Biotec) for 1 hour at 4° C. Labeled proteins were applied to a μMACS column placed in a μMACS separator (Miltenyi Biotec). Then, the column was washed three times with Wash Buffer 1 (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris-HCl, pH 8.0) and further washed once with 20 mM Tris-HCl, pH 7.5. The target proteins were eluted in 50 μl elution buffer preheated to 95° C (50 mM Tris-HCl, pH 6.8; 50 mM DTT; 1% SDS, 1 mM EDTA; 0.005% bromophenol blue; and 10% glycerol) for SDS-PAGE analysis.

Yang C.W., Hsieh M.H., Sun H.J, & Teng S.C. (2021). Nuclear envelope tethering inhibits the formation of ALT-associated PML bodies in ALT cells. Aging (Albany NY), 13(7), 10490-10516.

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Immunoprecipitation Using μMACS Separator

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For immunoprecipitation, we purchased μMACS™ Separator from Miltenyi Biotec (Germany) and performed following the manufacturer’s protocol with few changes. About 1 mg of protein was mixed with 1 μg monoclonal antibody and 50 μl μMACS Protein G MicroBeads (Miltenyi Biotec). After vigorous vortexing, the mixture was placed in ice for 30 min. To purify antibody-bound proteins, the mixture was applied to μ Columns (Miltenyi Biotec). The columns were primed with 200 μl 1X cell lysis buffer before applying the mixtures. Then, the column was washed with 200 μl high salt wash buffer (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 1 % NP-40 alternative, 4x) and 100 μl low salt wash buffer (20 mM Tris-HCl (pH 7.5)). For elution, we used 2X Laemmli Buffer (Bio-Rad) heated to 95 °C. The eluent was directly loaded on 4-15 % MiniPROTEAN^® TGX™ Precast Protein Gels for separation and western blotting analysis.

Kim S.M., Studnitzer B, & Esser-Kahn A. (2022). Heat Shock Protein (HSP) 90’s Mechanistic Role in Contact Hypersensitivity. Journal of immunology (Baltimore, Md. : 1950), 208(12), 2622-2631.

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Immunoprecipitation and Immunoblotting of Protein Complexes

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C33A or HeLa cells (2.5 × 10⁶) were seeded in 100 mm cell culture dishes. The next day, they were transfected; 48 h later, they were harvested; and then lysed in IP-buffer (50 mM HEPES pH 7.9, 150 mM NaCl, 0.3% (v/v) Igepal 630, 1 mM DTT, protease, and phosphatase inhibitors). Immunoprecipitation was carried out using magnetic anti-HA-beads (Miltenyi Biotech). Beads were washed with IP-buffer using μMACS columns and μMACS Separator (Miltenyi Biotech). Bound proteins were eluted in elution buffer [50 mM Tris HCl (pH 6.8), 50 mM DTT, 1% SDS, 1 mM EDTA, 0.005% bromephenol blue, 10% glycerol] heated to 95°C and analyzed by immunoblotting.
Separated proteins were transferred onto a 0.22 mm nitrocellulose membrane (Protran) in 10 mM CAPS and 10% (v/v) methanol (pH 10.3). Membranes were blocked in 5% milk in PBS with 0.1% Tween20. Membranes were incubated overnight with the primary Abs at 4°C. The following primary antibodies were used at the indicated dilution: HA-Tag (Cell signaling, rabbit mAb, C29F4, #3724,1:1,000), sYFP (Clontech, mouse mAb, JL-8, from # 632381, 1:1,000), HDAC3 (abcam, rabbit mAb, ab76295, 1:1,000), and TBL1 (Santa Cruz BT, mouse mAb, H-3, sc-137006, 1:750). Secondary fluorescence-labeled Abs (1:15,000; LI-COR) were added for 1 h, and signals were detected using the Odyssey Fc Infrared Imaging System (LI-COR Biosciences).

Kuehner F., Wong M., Straub E., Doorbar J., Iftner T., Roden R.B, & Stubenrauch F. (2023). Mus musculus papillomavirus 1 E8^E2 represses expression of late protein E4 in basal-like keratinocytes via NCoR/SMRT-HDAC3 co-repressor complexes to enable wart formation in vivo. mBio, 14(4), e00696-23.

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Immunoprecipitation of p53, ATM, and ATR

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NSPCs were incubated with or without 20 μM PJ34 for 24 h. Cell lysates were prepared from 4.0 × 10⁶ cells using RIPA buffer (Nacalai Tesque). An antibody against p53 (2524, 1:500; Cell Signaling), ATM (ab2618, 1:500; Abcam), or ATR (sc-1887, 1:500; Santa Cruz Biotechnology) was added to the cell lysates, and μMACS Protein A/G MicroBeads (Miltenyi Biotec) were added to magnetically label the immune complexes. Magnetically labeled proteins were collected by using μ Columns and μMACS Separator (Miltenyi Biotec), and analyzed by immunoblotting.

Okuda A., Kurokawa S., Takehashi M., Maeda A., Fukuda K., Kubo Y., Nogusa H., Takatani-Nakase T., Okuda S., Ueda K, & Tanaka S. (2017). Poly(ADP-ribose) polymerase inhibitors activate the p53 signaling pathway in neural stem/progenitor cells. BMC Neuroscience, 18, 14.

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Immunoprecipitation and Mass Spectrometry

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Three x 10⁸ cells (NanoLC-MS/MS analysis) or 1.5 x 10⁷ cells (immunoblot analysis) were harvested and then lysed in IP-buffer (50mM HEPES pH7.5, 150mM NaCl, 10% (v/v) glycerol, 5mM EDTA, 1mM DTT, protease and phosphatase inhibitors or 50mM HEPES pH7.9, 150mM NaCl, 0.3% (v/v) Igepal 630, 1mM DTT, protease and phosphatase inhibitors). Immunoprecipitation was carried out using magnetic anti-HA-beads (Miltenyi Biotech). Beads were washed with IP-buffer using μMACS columns and μMACS Separator (Miltenyi Biotech). Bound proteins were eluted in 4 x SDS gel loading buffer (Carl Roth) heated to 95°C and an aliquot analyzed by HA-immunoblotting to monitor the efficiency of the IP.

Dreer M., Fertey J., van de Poel S., Straub E., Madlung J., Macek B., Iftner T, & Stubenrauch F. (2016). Interaction of NCOR/SMRT Repressor Complexes with Papillomavirus E8^E2C Proteins Inhibits Viral Replication. PLoS Pathogens, 12(4), e1005556.

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Isolation and Analysis of EV-Derived RNA

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Up to 2mL of EVs isolated by SEC from serum of C57BL/6J mice stereotaxically injected in the striatum with lentiviral vectors encoding for Cre construct were incubated with 25uL of CD9, CD63 or CD81 MicroBeads (Miltenyi Biotec) overnight at 4°C in a tube rotator in the absence of light. Equilibration buffer (100μL) was applied on top of a μColumn (Miltenyi Biotec) that was previously placed in the magnetic field of the μMACS Separator attached to the MACS MultiStand and rinsed 3 times with 100μL of Isolation Buffer. The magnetically labelled samples were applied to the column which was placed in a mMACS Separator (Miltenyi Biotec). The column was washed 4x with Isolation Buffer and then placed in 1.5mL tubes. The sample was eluted by adding 100μL RNA lysis buffer (Miltenyi Biotec) to the column and flushed out by firmly pushing the plunger into the column. Downstream isolation of EV-derived RNA was performed using Total RNA Purification Plus Kit (Norgen) and according to manufacturer’s instructions. cDNA synthesis for mRNA was performed with iScript cDNA Synthesis Kit (Bio-Rad) and RT-PCR was performed with the Sso Advanced SYBR Green Supermix Kit (Bio-Rad) using the StepOnePlus Real-Time PCR System (Applied Biosystems).

Rufino-Ramos D., Leandro K., Perdigão P.R., O’Brien K., Pinto M.M., Santana M.M., van Solinge T.S., Mahjoum S., Breakefield X.O., Breyne K, & de Almeida L.P. (2023). Extracellular communication between brain cells through functional transfer of Cre mRNA. bioRxiv.

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