Micro bio spin p 30 columns

Manufactured by Bio-Rad

Sourced in United States, Germany

The Micro Bio-Spin P-30 columns are size-exclusion chromatography columns designed for the purification and desalting of small biomolecules. These columns efficiently separate molecules based on their size, allowing for the removal of salts, buffers, and other small molecules from protein samples.

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25 protocols using micro bio spin p 30 columns

Ribosomal Particle Buffer Exchange and Analysis

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Ribosomal particles were first buffer-exchanged into 1 M ammonium acetate solution (pH 7.5) with 2.5 mM magnesium acetate through a 30 kDa MW cutoff Bio-Rad P-30 Micro Bio-Spin columns, following vendor recommendations. Next, samples containing ribosomal particles were diluted and either directly used for mass spectrometry analysis at a final concentration of ∼ 100 nM or additionally further diluted to ∼ 2-5 nM for mass photometry analysis. For Hs40S ribosomal particles, final solution contained 250 mM ammonium acetate (pH 7.5) and 2.5 mM magnesium acetate. For Hs60S, So70S, and Hs80S ribosomal particles, final solution contained 750 mM ammonium acetate (pH 7.5) and 5 mM magnesium acetate.
Folded HCV IRES RNA samples were buffer-exchanged into 150 mM ammonium acetate solution (pH 7.5) using a 30 kDa Bio-Rad P-30 Micro Bio-Spin columns. Next, 100 nM Hs40S were mixed with different folds molar excess (∼ 2× and 4×) of HCV IRES RNA in ammonium acetate solution (pH 7.5) and incubated for 5 min at 37°C.

Lai S.H., Tamara S, & Heck A.J. (2021). Single-particle mass analysis of intact ribosomes by mass photometry and Orbitrap-based charge detection mass spectrometry. iScience, 24(11), 103211.

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Cy5-Labeling of Histone Octamer

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Histone octamer containing H3-K36C^Cy5 was prepared by thiol modification under native conditions. Octamer containing unlabeled H3-K36C was refolded and purified as described above, but the unfolding and refolding buffers contained TCEP, instead of β-mercaptoethanol, as a reducing agent. Octamer (0.5 mg) was adjusted to a concentration of 1 mg/ml with refolding buffer. One vial of Cy5 maleimide was dissolved in 50 μl of anhydrous DMSO and then mixed dropwise with the octamer solution. Labeling reactions were carried out overnight at 2°C, protected from light. β-mercaptoethanol (Sigma-Aldrich, 101458612) was added to a labeling reaction at a 100-fold molar excess of the dye to quench any unreacted species. Excess dye was removed using Micro Bio-Spin P-30 Columns (Bio-Rad, 7326202), pre-equilibrated with refolding buffer. Octamer concentration and labeling efficiency were estimated spectrophotometrically from the absorbance measurement at 276 and 650 nm. Octamer was flash-frozen in liquid nitrogen and stored at −80°C.

Gruszka D.T., Xie S., Kimura H, & Yardimci H. (2020). Single-molecule imaging reveals control of parental histone recycling by free histones during DNA replication. Science Advances, 6(38), eabc0330.

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Gemcitabine-modified Aptamer Synthesis

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Gemcitabine-5′-triphosphate (dFdCTP) was purchased from Sierra Bioresearch. 5-F-2′-UTP triphosphate (5FdUTP) was purchased from TriLink Biotechnologies. DuraScribe T7 transcription kit (Epicenter Biotechnologies) was used to incorporate dFdCTP and 5FdUTP into aptamers. Micro Bio-spin P30 columns (Bio-Rad) were used to remove unincorporated dFdCTP and 5FdUTP. Primary antibodies for γ-H2AX were purchased from Cell Signaling Technology. Secondary antibodies conjugated with Alexa 88 were purchased from Thermo Fisher Scientific.

Yoon S., Huang K.W., Reebye V., Spalding D., Przytycka T.M., Wang Y., Swiderski P., Li L., Armstrong B., Reccia I., Zacharoulis D., Dimas K., Kusano T., Shively J., Habib N, & Rossi J.J. (2016). Aptamer-Drug Conjugates of Active Metabolites of Nucleoside Analogs and Cytotoxic Agents Inhibit Pancreatic Tumor Cell Growth. Molecular Therapy. Nucleic Acids, 6, 80-88.

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Multimodal Imaging of Vertebrate Development

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The following constructs were used in this study: pGF-CMV-GCaMP6s (Chen et al., 2013 (link)), pBH-R4/R2 (Heim et al., 2014 (link)), p5E-bactin2, pCS2FA-transposase (Kwan et al., 2007 ), p5E-ubi (Mosimann et al., 2011 (link)), pCS2+wnt5b (Lin et al., 2010 (link)), and pCS2+cyc/ndr2 (Rebagliati et al., 1998 (link)). The GCaMP6s open reading frame (ORF) was amplified from pGF-CMV-GCaMP6s using Phusion High-Fidelity DNA polymerase (Invitrogen) with GCaMP6s-Forward and -Reverse primers (Table S1). The amplified GCaMP6s ORF was cloned into pENTR/D-TOPO (Invitrogen) and sequenced. To generate Tol2 destination constructs, p5E-βactin2 or p5E-ubi promoter sequences were recombined with pENTR/D-GCaMP6s into pBH-R4/R2 using the Gateway LR Clonase II Plus enzyme (Invitrogen).
To generate cyc/ndr2 RNA, pCS2+cyc/ndr2 plasmid was linearized with Asp718I, purified (Qiagen), and used as a template for RNA synthesis with the mMessage mMachine SP6 kit (Ambion). The resulting RNA was purified with the Micro Bio-Spin P-30 columns (Bio-Rad). 50 pg cyc/ndr-2 RNA mixed with 0.1% Texas Red was injected at 64-cell stage to 1–2 marginal blastomeres, or 400 pg was injected at 512-cell stage to the yolk cell. pCS2+wnt5b was linearized with ApaI, and transcribed and purified as described above. 50–100 pg wnt5b synthetic RNA was injected at 1-cell stage.

Chen J., Xia L., Bruchas M.R, & Solnica-Krezel L. (2017). Imaging early embryonic calcium activity with GCaMP6s transgenic zebrafish. Developmental biology, 430(2), 385-396.

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Quantifying Intracellular dsRNA Uptake

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DsRNA was labeled with Alexafluor 488 using the Ulysis nucleic acid labeling kit (Invitrogen). Excess labeling reagent was removed using Micro Biospin P-30 columns (BioRad, Hercules, CA). A549 cells were seeded and grown to confluence in 96 well plates and serum starved for 12-16 h. The next day, cells were pretreated with wortmannin for 1h at 37°C prior to addition of fluorescently labeled dsRNA. After one hour, unbound dsRNA was removed by washing cells with PBS. Total fluorescence was measured using the fluorescence plate reader (SpectraMax i3) prior to removal of unbound dsRNA. 0.025% tryphan blue was subsequently added to quench the extracellular fluorescence signal, thereby measuring the intracellular fluorescence. Results were reported as a percentage of total fluorescence relative to control cells.

Nellimarla S., Baid K., Loo Y.M., Gale M J.r., Bowdish D.M, & Mossman K.L. (2015). Class A scavenger receptor-mediated dsRNA internalization is independent of innate antiviral signaling and does not require PI3K activity. Journal of immunology (Baltimore, Md. : 1950), 195(8), 3858-3865.

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Synthesis of Capped and Uncapped RNA

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PCR reactions were performed with DreamTaq (Thermo Fisher) in 50 μL reaction volume using 10 ng of a pYes plasmid construct as a template, 0.4 μM forward (5′-CGGATCGGACTACTAGCAGCTG -3′) and 0.4 μM reverse (5′-TTCATTAATGCAGGGCCGCAAATT-3′) primers that anneal upstream and downstream of the P_T7 and the TER_CYC1 elements, respectively. PCR products were purified and concentrated using type D4004 ZYMO column (ZYMO Research) and DNA concentrations were estimated spectrophotometrically. To synthetize uncapped RNA, 1 μg of PCR-generated DNA template was transcribed in the T7-polymerase reaction using TranscriptAid T7 High Yield Transcription kit (Thermo Scientific) according to the manufacturer's recommendations. Reaction mixtures were incubated for 120 min at 37 °C, followed by DNase treatment for 15 min at 37 °C. RNA transcripts were first purified by phenol/chloroform extraction, followed by EtOH precipitation; pellets were resuspended in 50 μL of H₂O and separated from unincorporated nucleotides by gel filtration on Micro Bio-Spin P-30 columns (Bio-Rad). RNA was aliquoted and stored at − 80 °C.
To generate capped RNA, purified PCR products (described above) were used as DNA templates in coupled T7 RNA polymerization–RNA capping reactions (mMESSAGE mMACHINE kit, Thermo Fisher #AM1344M), incubated for 1 h at 37 °C.

Trainor B.M., Ghosh A., Pestov D.G., Hellen C.U, & Shcherbik N. (2021). A translation enhancer element from black beetle virus engages yeast eIF4G1 to drive cap-independent translation initiation. Scientific Reports, 11, 2461.

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Ion Mobility Mass Spectrometry of Biomolecules

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Samples were buffer exchanged into 200 mM ammonium acetate using Micro Bio-Spin P-30 columns (Bio-Rad, Hercules, CA) at an initial concentration of ~1 μM. The final concentrations of the samples ranged from 100 to 900 nM, based on expected losses during buffer exchange. We performed IM-MS experiments on a Synapt G2 IM-MS platform (Waters Corp., Milford, MA) equipped with a nESI source. Briefly, the capillary voltage was set to 1.5 kV, with sampling and extraction cone voltages set to 0 V to preserve noncovalent interactions. The trap and transfer collision energies were both set to 4 V. Optimal mobility parameters were as previously published (Zhong et al., 2011 (link)), with IM gas pressure set at approximately ~4 mBar with the wave height and wave velocity set to 15 V and 150 m/s, respectively. Data were processed using Masslynx and Driftscope (Waters Corp., Milford, MA). Mass assignments were calculated using the maximum entropy method as implemented in ESIprot (Winkler, 2010 (link)).

Eschweiler J.D., Farrugia M.A., Dixit S.M., Hausinger R.P, & Ruotolo B.T. (2018). A Structural Model of the Urease Activation Complex Derived from Ion Mobility-Mass Spectrometry and Integrative Modeling. Structure (London, England : 1993), 26(4), 599-606.e3.

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Aptamer Synthesis with 2F′-modified Pyrimidines

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The DuraScribe T7 transcription kit (Lucigen, Middleton, WI, USA) was used to incorporate 2F′-modified pyrimidines into aptamers. Micro Bio-spin P30 columns (Bio-Rad, Hercules, CA, USA) were used to remove unincorporated nucleotide triphosphates (NTPs).

Yoon S., Wu X., Armstrong B., Habib N, & Rossi J.J. (2018). An RNA Aptamer Targeting the Receptor Tyrosine Kinase PDGFRα Induces Anti-tumor Effects through STAT3 and p53 in Glioblastoma. Molecular Therapy. Nucleic Acids, 14, 131-141.

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Quantifying ABCE1-mediated ATP occlusion

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The occlusion of ATP and ADP by ABCE1 was determined by using ³²P-α-ATP (Hartmann Analytics, 222 TBq/mmol, 370 MBq/ml) and the analysis as already described for the ATPase assay. Here, 9 μM cold ATP was supplemented 1:500 with radioactive tracer, and final concentrations of 0.6 μM ATP and 0.3 μM ABCE1 were incubated for 30 s at 45 °C in TrB25. Samples were then quickly chilled on ice and supplemented with 0.5 mM cold ATP to reduce unspecific binding. To determine the intensity of the load, 1 μl sample was directly spotted onto the TLC plate. ABCE1 and occluded ATP molecules were separated from residual ATP by SEC in Micro Bio-Spin™ P30 columns (Biorad). 1 μl of the eluted sample were used for TLC analysis. The signals for ATP and ADP in the load samples summed up to a total corresponding to 0.6 μM of ATP. Retention of ABCE1 by the SpinColumn was calculated using SDS-PAGE analysis. An example of the calculation procedure is given in Figure S8. Nucleotide occlusion was preformed twice, bars in Figure 4B represent a mean ± SD value and the radiogram in Figure 4A is representative for both independent experiments.

Nürenberg-Goloub E., Heinemann H., Gerovac M, & Tampé R. (2018). Ribosome recycling is coordinated by processive events in two asymmetric ATP sites of ABCE1. Life Science Alliance, 1(3), e201800095.

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Radiolabeling of Modified DNA Substrates

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The oligodeoxynucleotide substrates containing a single εA (40-mer, 5′-d(GCTACCTACCTAGCGACCTεACGACTGTCCCACTGCT-CGAA)-3′, Eurogentec S.A., Belgium), εC (25-mer, 5′-d(ATTCTCGTT-AGGATεCGCGTCAAGCC)-3′, Chemgenes Corporation, USA), or 1,N²-εG (18-mer, 5′-(TCACεGAATCCTTACGAGCCCCC)-3′, [60 (link),61 (link)] were ³²P-labeled at the 5′ end by T4 polynucleotide kinase (Takara) with an excess of [γ-³²P]ATP (3000 Ci/mmol, Hartmann Analytic). Radiolabeled oligodeoxynucleotides were purified from unincorporated radioactivity using Micro Bio-Spin P-30 columns (Bio-Rad) according to the manufacturer’s instructions. The ³²P-labeled oligodeoxynucleotides, when necessary, were annealed to their complementary oligonucleotides (present in a 2-fold molar excess) by incubation for 2 min at 90 °C and subsequent cooling to room temperature. Formation of duplexes was verified by nondenaturing PAGE.

Zdżalik D., Domańska A., Prorok P., Kosicki K., van den Born E., Falnes P.Ø., Rizzo C.J., Guengerich F.P, & Tudek B. (2015). Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins. DNA repair, 30, 1-10.

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