Cell pellets were resuspended in “lysis buffer” (50mM Tris-HCl, 150mM NaCl, 1% NP-40, Pierce Protease and Phosphatase Inhibitor Mini Tablets (cat#A32959) in purified water at pH 7.4) and homogenized before incubating on ice for 20 min. Lysates were centrifuged at 16,000 × g for 30 min at 4 °C and the supernatant was normalized to 2 mg/mL in lysis buffer. 250 μL of soluble lysate fraction was incubated with 50 μL of μMACS™ DYKDDDDK Isolation Kit anti-FLAG beads for 30 minutes at 4 °C. The lysate-bead mix was added to a μColumn (Miltenyi Biotec) that had been prewashed with lysis buffer and was attached to a magnetic MACS MultiStand. Beads were washed three times with lysis buffer and five times with 50 mM Tris-HCl/150 mM NaCl in water (all at 4 °C) and incubated with 20 μL of boiling elution buffer (from kit) for five minutes followed by addition of 50 μL of boiling elution buffer and elution (this elution was repeated twice); all elution fractions were combined.
μcolumn
Manufactured by Miltenyi Biotec
The μColumn is a compact and versatile magnetic separation column designed for use in small-scale cell separation procedures. It facilitates the isolation and purification of target cells from a heterogeneous cell population using magnetic beads or nanoparticles. The μColumn is suitable for a wide range of applications, including immunomagnetic cell separation, stem cell isolation, and sample preparation for downstream analysis.
Automatically generated - may contain errors
Lab products found in correlation
Infinite m1000 pro plate reader, by Tecan (2 mentions)
Qubit rna hs assay kit, by Thermo Fisher Scientific (2 mentions)
μmacs streptavidin microbeads, by Miltenyi Biotec (2 mentions)
Minielute column, by Zymo Research (2 mentions)
Epiquik m6a rna methylation quantification kit, by Epigentek (2 mentions)
μmacs gfp tagged protein isolation kit, by Miltenyi Biotec (2 mentions)
Ssoadvanced sybr green supermix kit, by Bio-Rad (1 mentions)
μmacs separator, by Miltenyi Biotec (1 mentions)
Total rna purification plus kit, by Norgen Biotek (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
6 protocols using μcolumn
1
Efficient Affinity-Based Protein Purification
2
Quantification of m6A RNA Methylation
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Total RNA from E. coli Tuner (DE3) cells was purified as described above and adjusted to a volume of 200 μL. Total RNA was denatured at 75 °C for 5 min and incubated at 42 °C for 10 min with 1 μg of dual biotin labeled DNA probe complimentary to a unique 30 base region within the synthetic target. Probe annealed target RNA was bound to 100 μL of μMACS Streptavidin microbeads (Miltenyi Biotec) and incubated at 4 °C for 15 min. Magnetic separation was done with a μColumn (Miltenyi Biotec). RNA:probe was passed over the μColumn and washed twice with 150 uL TE buffer. Probe bound RNA was eluted by adding 150 μL of H2O warmed to 90 °C. Eluted RNA was purified and concentrated using a MiniElute column (Zymo Research). Resulting RNA concentration was determined using a Qubit RNA HS assay kit (Thermo Fisher). RNA was serial diluted and m6A was quantified with the EpiQuik m6A RNA methylation quantification kit (Epigentek) according to manufacturer’s protocol. Flourometric m6A signal was measured on an Infinite Pro M1000 plate reader (Tecan) at 530EX/590EM nm.
3
Quantification of m6A RNA Methylation
4
Isolation of lipid droplets using Co-IP
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The Co-IP LD isolation method was performed using a μMACS GFP-tagged protein isolation kit (Miltenyi Biotec, Gladbach, Germany) following the method of a previous study (Shimada et al., 2014 (link)). Four-week-old hise1-2/pLDAP3:LDAP3-GFP plants (0.5 g fresh weight) and wild-type plants expressing cytosolic GFP alone were ground with 1.5 mL extraction buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM CaCl2, and 1 mM MgCl2 using a pestle and mortar. The extracts were centrifuged at 1,000 g for 5 min at 4°C followed by 8,000 g for 10 min at 4°C. The supernatants (1 mL each) were subjected to immunoprecipitation with 50 μL of anti-GFP microbeads (μMACS GFP-tagged protein isolation kit). The samples were incubated at 4°C for 30 min and applied to a μ column (Miltenyi Biotec). The column was washed with 1 mL of the extraction buffer. Pure immunoprecipitates were eluted with 50 μL of sample buffer (100 mM Tris-HCl pH 6.8, 4% [w/v] SDS, 12% [v/v] 2-mercaptoethanol, and 20% [v/v] glycerol) and defined as isolated LDs.
5
Co-Immunoprecipitation of GFP-Tagged Proteins
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Co-IP assays were performed using a μMACS GFP-tagged protein isolation kit (Miltenyi Biotec). Three-week-old A. thaliana plants (0.5 g fresh weight) expressing MYOB2-GFP or MYOB14-GFP were ground in 1 mL of lysis buffer (150 mM NaCl, 1% [v/v] Ecosurf EH-9, and 50 mM Tris-HCl pH 8.0). The extracts were centrifuged at 1,000 × g for 1 min at 4°C. This was followed by centrifugation of the supernatants at 15,000 × g for 10 min at 4°C. A 1-mL aliquot of each supernatant was subjected to Co-IP with 50 μL of anti-GFP microbeads. Samples were incubated at 4°C for 30 min and applied to a μ column (Miltenyi Biotec). The column was washed with 1 mL of lysis buffer and 0.2 mL of 20 mM Tris-HCl, pH 7.5. Pure immunoprecipitates were eluted with 50 μL of sample buffer (100 mM Tris-HCl pH 6.8, 4% [w/v] SDS, 12% [v/v] 2-mercaptoethanol, and 20% [v/v] glycerol).
6
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).
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