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Microspin column

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Microspin columns are compact, single-use centrifugation devices designed to perform rapid and efficient separations of biomolecules such as proteins, nucleic acids, and other macromolecules. These columns contain a specialized resin or membrane that selectively retains or elutes the target analyte during the centrifugation process. Microspin columns offer a convenient and reproducible way to purify or desalt samples in a variety of laboratory applications.

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Lab products found in correlation

Complete protease inhibitor, by Roche (3 mentions) Anti nek1 h3t1, by Merck Group (2 mentions) Anti flag, by Merck Group (2 mentions) Protein a g plus agarose bead, by Santa Cruz Biotechnology (2 mentions) Complete protease inhibitor cocktail, by Roche (2 mentions) Phosstop tablet, by Roche (1 mentions) Millex gp 0.22 m, by Merck Group (1 mentions) Amplitaq gold, by Roche (1 mentions) Kod fx, by Toyobo (1 mentions) Adenosine triphosphate (atp), by Thermo Fisher Scientific (1 mentions) T4 polynucleotide kinase, by New England Biolabs (1 mentions) Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)

Close spelling variants of this product

Illustra G-25 MicroSpin columns Ilustra MicroSpin G-25 columns MicroSpin S-300 HR columns S400-HR microspin columns Illustra microspin columns Illustra MicroSpin S-200 HR columns G-50 microspin columns Illustra G-50 microspin columns G25 Microspin columns MicroSpin S-400 columns

10 protocols using microspin column

1

Rab13 Activation Assay Protocol

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The Rab13 activation assay was previously described (Wall et al., 2017 (link)). In brief, a GST fusion protein of the
RBD of OCRL (amino acids 539–901) was used to pull down active GTP-loaded
Rab13 from a stable cell line expressing myc-tagged Rab13. MicroSpin columns
(27-3565-01; GE Healthcare) were used for all of the pulldowns. Beads were
washed with ice-cold lysis buffer (20 mM Tris-HCl, pH 7.2, 150 mM NaCl, 5 mM
MgCl2, 1% NP-40, and 5% glycerol); cOmplete protease inhibitors
(Roche Applied Science) and phosSTOP tablets (Roche Applied Science) were added
to lysis buffer before use. Elution was achieved conventionally by boiling in
2× SDS-PAGE sample buffer for 5 min. The samples were subjected to
immunoblots. Total and active Rab13 is detected using anti-myc antibody from
input and pulldown extracts, respectively.
Condon N.D., Heddleston J.M., Chew T.L., Luo L., McPherson P.S., Ioannou M.S., Hodgson L., Stow J.L, & Wall A.A. (2018). Macropinosome formation by tent pole ruffling in macrophages. The Journal of Cell Biology, 217(11), 3873-3885.
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2

Triplex Displacement Assay Protocol

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The substrate of the triplex displacement assay had two components: A Triplex Forming Oligo (TFO), 100 nM of which was labelled at the 5′ end using T4 polynucleotide kinase in the presence of 32P-γATP at 37°C for 30 min. Post the incubation, the reaction was stopped by heat inactivating T4 PNK at 65°C for 20 min. MicroSpin columns (GE) was used for purifying the TFO and stored at −30°C for future use. The second component was a plasmid DNA consisting of an enzyme binding cassette and a triplex binding site (TBS) 604 bp downstream of the enzyme binding cassette. Methylation and linearization of the plasmid were done with a similar protocol to the plasmids used for cleavage assays. To form the triplex, 50 nM of the linearized plasmid was incubated along with 25 nM TFO, 10 mM MES (pH 5.5) and 200 mM MgCl2 at 57°C for 15 min. Subsequently, the reaction was allowed to cool to 20°C and kept overnight. The triplex displacement reaction was carried out at 20°C at different time points by incubating SauUSIH119A with the triplex DNA for 2 minutes following which cold TFO and ATP were added. The curve (% of triplex displaced versus time in minutes) was fit using the one-phase association function in GraphPad Prism. The equation used was: Y = Ymax1(1 – e−K.X). The error bars represent the standard error of the mean across three independent experiments.
Tumuluri V.S., Rajgor V., Xu S.Y., Chouhan O.P, & Saikrishnan K. (2021). Mechanism of DNA cleavage by the endonuclease SauUSI: a major barrier to horizontal gene transfer and antibiotic resistance in Staphylococcus aureus. Nucleic Acids Research, 49(4), 2161-2178.
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3

Affinity Purification of NEK1 Protein Complexes

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Protein A/G PLUS-agarose beads (Santa Cruz Biotechnology) were washed three times in extraction buffer (10mM HEPES [pH7.9], 10mM NaCl, 3mM MgCl2, freshly added 1mM DTT, 1mM Na3VO4) supplemented with complete protease inhibitor cocktail (Roche) and then coupled to polyclonal anti-NEK1 (H3T1) or polyclonal anti-FLAG (Sigma, F7425) overnight at 4°C. Coupled beads were washed three times in extraction buffer and incubated with fresh bovine retinal lysates overnight at 4°C. Retinas were homogenized by sonication on ice two times for 30 seconds in extraction buffer (10mM HEPES [pH7.9], 10mM NaCl, 3mM MgCl2, freshly added 1mM DTT, 1mM Na3VO4) supplemented with complete protease inhibitor cocktail (Roche). Beads were washed five times with extraction buffer, transferred to microspin columns (GE Healthcare) and washed three times in washing buffer (1xTBS, 0.12% NP-40, supplemented with complete protease inhibitors (Roche)). Then, neutralization buffer (1M Tris/HCl, [pH8.0]) was added. Eluates were retrieved by adding 3x elution buffer (200 mM glycine [pH2.5]) for 20 min. at 4°C and subjected to protein precipitation with chloroform and methanol. Protein precipitates were subsequently subjected to mass spectrometry analysis and peptide identification as previously described48 (link).
Wheway G., Schmidts M., Mans D.A., Szymanska K., Nguyen T.M., Racher H., Phelps I.G., Toedt G., Kennedy J., Wunderlich K.A., Sorusch N., Abdelhamed Z.A., Natarajan S., Herridge W., van Reeuwijk J., Horn N., Boldt K., Parry D.A., Letteboer S.J., Roosing S., Adams M., Bell S.M., Bond J., Higgins J., Morrison E.E., Tomlinson D.C., Slaats G.G., van Dam T.J., Huang L., Kessler K., Giessl A., Logan C.V., Boyle E.A., Shendure J., Anazi S., Aldahmesh M., Al Hazzaa S., Hegele R.A., Ober C., Frosk P., Mhanni A.A., Chodirker B.N., Chudley A.E., Lamont R., Bernier F.P., Beaulieu C.L., Gordon P., Pon R.T., Donahue C., Barkovich A.J., Wolf L., Toomes C., Thiel C.T., Boycott K.M., McKibbin M., Inglehearn C.F., Stewart F., Omran H., Huynen M.A., Sergouniotis P.I., Alkuraya F.S., Parboosingh J.S., Innes A.M., Willoughby C.E., Giles R.H., Webster A.R., Ueffing M., Blacque O., Gleeson J.G., Wolfrum U., Beales P.L., Gibson T., Doherty D., Mitchison H.M., Roepman R, & Johnson C.A. (2015). An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes. Nature cell biology, 17(8), 1074-1087.
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4

Co-immunoprecipitation of PSPC1 from Testis Lysates

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Testis lysates prepared as for co-IPs were precleared with GS4B slurry (60 μl/200 μl lysate, 4°C with rotation, ∼2 h). GS4B slurry aliquots (60 μl) were equilibrated with transport buffer (20 mM HEPES-NaOH, pH 7.3, 110 mM CH3COOK, 2 mM Mg(CH3COO)2, 5 mM CH3COONa, 0.5 mM EGTA, 2 mM DTT, PIC 1:500) in microspin columns (GE Life Sciences), and then GST-tagged protein (300 pmol) was added in 300 μl of transport buffer for binding to GS4B (4°C, 1 h with rotary agitation). Precleared testis lysate (200 μl) was added and incubated O/N (4°C, gentle rotation). Flowthrough, washes (transport buffer), and eluate (2× sample buffer) samples were collected for Western blot detection of PSPC1.
Major A.T., Hogarth C.A., Miyamoto Y., Sarraj M.A., Smith C.L., Koopman P., Kurihara Y., Jans D.A, & Loveland K.L. (2015). Specific interaction with the nuclear transporter importin α2 can modulate paraspeckle protein 1 delivery to nuclear paraspeckles. Molecular Biology of the Cell, 26(8), 1543-1558.
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5

Affinity Purification of TAP-Tagged Proteins

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For the interaction analysis, we used HEK293T cells transfected with Fltp-TAP-TAG and as a control HEK293T cells only transfected with TAP-TAG.
The cells were rinsed with warm PBS and lysed with 1 ml ice cold lysis buffer (50 mM Tris/HCl, pH 7.4, 150 mM sodium chloride, 2 mM EDTA, pH 8, 1% Nonidet P-40, filtrate sterile) per 10 cm dish on ice. Cells were spinned down for 10 min at 10000×g at 4°C. Lysates were cleaned by filtration through syringe filters (MILLEX GP, 0.22 µm, Millipore). 1 mg of the filtered lysate was incubated with 50 µl Strep-Tactin superflow resin (IBA) in 1 ml of lysis buffer for 1 hr at 4°C in an overhead tumbler. The protein–Strep-Tactin solution was spinned down for 30 s at 7000×g and transferred to microspin columns (GE-Healthcare). The supernatant was spinned down for 5 s at 100×g, washed three times with 500 µl TBST (100 mM Tris/HCl, pH 7.4, 1.5 M sodium chloride, 1.0% Tween20), and centrifuged for 5 s, 100×g. The protein was eluted by 500 µl desthiobiotin elution buffer (IBA) and incubated for 10 min while mixing the resin for several times. The elute can now be used for western blot analysis.
Gegg M., Böttcher A., Burtscher I., Hasenoeder S., Van Campenhout C., Aichler M., Walch A., Grant S.G, & Lickert H. (2014). Flattop regulates basal body docking and positioning in mono- and multiciliated cells. eLife, 3, e03842.
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6

HHV-6B Genome Sequencing Protocol

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To reveal the contents of the HHV genome in the HUV-EC-C genome, PCR was performed on 15 regions (Fig. 1) using AmpliTaq Gold (Roche Indianapolis, IN, USA) or KOD FX (TOYOBO, Osaka, Japan) DNA polymerase. Primers were designed based on the HHV-6B HST reference sequence using a gene analysis software, Genetyx, listed in Table S2. The PCR products were run on a 2% agarose gel. A single band of the PCR products was purified by MicroSpin Columns (GE Healthcare, Tokyo, Japan) and analyzed by standard Sanger sequencing. Sequence homology of these PCR products with HHV-6B strains HST (AB021506.1), Z29 (AF157706.1) and HHV-6A U1102 (X83413.1) were analyzed by Genetyx.

Primer positions on HHV-6B genome DNA. Each primer pair is indicated by arrows. Primer sequences are listed in Table S2

Shioda S., Kasai F., Ozawa M., Hirayama N., Satoh M., Kameoka Y., Watanabe K., Shimizu N., Tang H., Mori Y, & Kohara A. (2017). The human vascular endothelial cell line HUV-EC-C harbors the integrated HHV-6B genome which remains stable in long term culture. Cytotechnology, 70(1), 141-152.
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7

Affinity Purification of NEK1 Protein Complexes

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Protein A/G PLUS-agarose beads (Santa Cruz Biotechnology) were washed three times in extraction buffer (10mM HEPES [pH7.9], 10mM NaCl, 3mM MgCl2, freshly added 1mM DTT, 1mM Na3VO4) supplemented with complete protease inhibitor cocktail (Roche) and then coupled to polyclonal anti-NEK1 (H3T1) or polyclonal anti-FLAG (Sigma, F7425) overnight at 4°C. Coupled beads were washed three times in extraction buffer and incubated with fresh bovine retinal lysates overnight at 4°C. Retinas were homogenized by sonication on ice two times for 30 seconds in extraction buffer (10mM HEPES [pH7.9], 10mM NaCl, 3mM MgCl2, freshly added 1mM DTT, 1mM Na3VO4) supplemented with complete protease inhibitor cocktail (Roche). Beads were washed five times with extraction buffer, transferred to microspin columns (GE Healthcare) and washed three times in washing buffer (1xTBS, 0.12% NP-40, supplemented with complete protease inhibitors (Roche)). Then, neutralization buffer (1M Tris/HCl, [pH8.0]) was added. Eluates were retrieved by adding 3x elution buffer (200 mM glycine [pH2.5]) for 20 min. at 4°C and subjected to protein precipitation with chloroform and methanol. Protein precipitates were subsequently subjected to mass spectrometry analysis and peptide identification as previously described48 (link).
Wheway G., Schmidts M., Mans D.A., Szymanska K., Nguyen T.M., Racher H., Phelps I.G., Toedt G., Kennedy J., Wunderlich K.A., Sorusch N., Abdelhamed Z.A., Natarajan S., Herridge W., van Reeuwijk J., Horn N., Boldt K., Parry D.A., Letteboer S.J., Roosing S., Adams M., Bell S.M., Bond J., Higgins J., Morrison E.E., Tomlinson D.C., Slaats G.G., van Dam T.J., Huang L., Kessler K., Giessl A., Logan C.V., Boyle E.A., Shendure J., Anazi S., Aldahmesh M., Al Hazzaa S., Hegele R.A., Ober C., Frosk P., Mhanni A.A., Chodirker B.N., Chudley A.E., Lamont R., Bernier F.P., Beaulieu C.L., Gordon P., Pon R.T., Donahue C., Barkovich A.J., Wolf L., Toomes C., Thiel C.T., Boycott K.M., McKibbin M., Inglehearn C.F., Stewart F., Omran H., Huynen M.A., Sergouniotis P.I., Alkuraya F.S., Parboosingh J.S., Innes A.M., Willoughby C.E., Giles R.H., Webster A.R., Ueffing M., Blacque O., Gleeson J.G., Wolfrum U., Beales P.L., Gibson T., Doherty D., Mitchison H.M., Roepman R, & Johnson C.A. (2015). An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes. Nature cell biology, 17(8), 1074-1087.
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8

Phosphorylation and Dephosphorylation of RNA Pol II

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200 ng of GST-CTD (c-terminal domain of RNA Pol II) was incubated with CDK9/CyclinT1 (Abcam, Cambridge, MA, USA) for 2 hours at 30° C in kinase buffer containing 40 mM HEPES, pH 7.5, 10 mM MgCl2, 5 mM dithiothreitol, and 500 μM ATP (Invitrogen). Reaction was stopped by removing unincorporated ATP through a MicroSpin Column (GE Healthcare) according to the manufacturer’s protocol. PTEN phosphatase activity was tested by incubating phosphorylated GST-CTD with PTEN and Ssu72 (a positive control) in phosphatase buffer containing 50mM Tris-HCl, pH 6.5, 10 mM MgCl2, 20 mM KCl, and 5 mM DTT. After 2 hours of incubation at 30° C, reactions were stopped by adding 2× Laemmli buffer and incubated at 100° C for 5 minutes before immunoblotting.
Abbas A., Romigh T, & Eng C. (2019). PTEN interacts with RNA polymerase II to dephosphorylate polymerase II C-terminal domain. Oncotarget, 10(48), 4951-4959.
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9

Radioisotope Labeling of dsDNA

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dsDNA substrate 100 nM was labeled at 5′-end with T4 polynucleotide kinase (New England Biolabs) in presence of (gamma-32P ATP) at 37°C for 30 min. Polynucleotide kinase was heat inactivated at 65°C for 20 min. The DNA was purified using MicroSpin column (GE Healthcare) and stored at –30°C until further use.
Ahmad I., Kulkarni M., Gopinath A, & Saikrishnan K. (2018). Single-site DNA cleavage by Type III restriction endonuclease requires a site-bound enzyme and a trans-acting enzyme that are ATPase-activated. Nucleic Acids Research, 46(12), 6229-6237.
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10

Immunoprecipitation of Importin Proteins

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Adult mouse testis were homogenized on ice through needles (18 to 30 gauge) in IP lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X‑100, PIC 1:500) and then centrifuged. Lysate supernatant (200 μl [∼200 μg protein]) was incubated (4°C with rotation, 2 h) with 2.0 μg anti-IMPα2 (cat. no. sc6917, lot no. c1407; Santa Cruz Biotechnology) and added to a microspin column (GE Life Sciences) containing 60 μl prewashed protein A/G-PLUS-Agarose (Santa Cruz Biotechnology, distributed by ThermoFisher Scientific, Scoresby, Australia), and the volume was brought to 500 μl with IP lysis buffer for O/N incubation (4°C with rotation). Samples of flowthrough, washes (IP lysis buffer), and elution in 2× sample buffer (4% vol/vol SDS, 25% vol/vol glycerol, 12.5% vol/vol [0.5M Tris HCl, pH 6.8], 5% vol/vol β-mercaptoethanol, bromophenol blue) were collected for Western blot detection of PSPC1 and IMPβ1.
Major A.T., Hogarth C.A., Miyamoto Y., Sarraj M.A., Smith C.L., Koopman P., Kurihara Y., Jans D.A, & Loveland K.L. (2015). Specific interaction with the nuclear transporter importin α2 can modulate paraspeckle protein 1 delivery to nuclear paraspeckles. Molecular Biology of the Cell, 26(8), 1543-1558.
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