Bio spin 6 column

Manufactured by Bio-Rad

Sourced in United States, United Kingdom, Spain

The Bio-Spin 6 columns are size-exclusion chromatography columns designed for rapid purification and desalting of biomolecules. They are used to separate analytes based on their size and molecular weight.

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Lab products found in correlation

Masslynx software, by Waters Corporation (9 mentions) Synapt g2 si hdms mass spectrometer, by Waters Corporation (7 mentions) Q exactive mass spectrometer, by Thermo Fisher Scientific (4 mentions) Xcalibur 2, by Thermo Fisher Scientific (4 mentions) Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions) Anti flag agarose beads clone m2, by Merck Group (3 mentions) Phosstop, by Merck Group (3 mentions) Flag peptide, by Merck Group (3 mentions) Q exactive uhmr mass spectrometer, by Thermo Fisher Scientific (3 mentions) γ 32p atp, by PerkinElmer (3 mentions) Masslynx 4, by Waters Corporation (3 mentions) 211 mouse monoclonal antibody mab, by Santa Cruz Biotechnology (3 mentions) Thermomixer, by Eppendorf (3 mentions) Ammonium acetate, by Merck Group (2 mentions) 4 maleimido benzophenone, by Santa Cruz Biotechnology (2 mentions) T4 polynucleotide kinase, by New England Biolabs (2 mentions) Mmessage mmachine t7 transcription kit, by Thermo Fisher Scientific (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions) Dntps, by Meridian Bioscience (2 mentions) Es380 borosilicate nanoes spray emitters, by Thermo Fisher Scientific (2 mentions)

101 protocols using bio spin 6 column

Characterizing TcpF-TcpB Interactions

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The buffers for TcpF, the TcpF-Y5A mutant, the TcpF-L100D mutant, and TcpB were exchanged into 200 mM ammonium acetate (pH 7; Sigma-Aldrich) by passing the proteins through a Bio-Spin 6 column (Bio-Rad, 10-kDa cutoff). Buffer-exchanged TcpF or its mutants were mixed with TcpB and incubated at room temperature for 20 min to obtain mixtures of 20 μM TcpF/5 μM TcpB, 20 μM TcpF-Y5A/5 μM TcpB, 50 μM TcpF-Y5A/5 μM TcpB, and 20 μM TcpF-L100D/5 μM TcpB. These mixtures, 20 μM TcpF and 20 μM TcpF mutants, were analyzed by nano-electrospray ionization mass spectrometry with gold glass capillaries made in-house (5-μl sample loaded per analysis). Spectra were recorded on Q Exactive UHMR Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific) in positive ionization mode at 1.2-kV spray voltage with 21.0-V source DC offset, 40-V hollow cathode discharge (HCD) voltage, and 5.0 trapping gas setting. The spectra were calibrated using cesium iodide (4 mg/ml) and analyzed using BioPharma Finder software (Thermo Fisher Scientific).

Oki H., Kawahara K., Iimori M., Imoto Y., Nishiumi H., Maruno T., Uchiyama S., Muroga Y., Yoshida A., Yoshida T., Ohkubo T., Matsuda S., Iida T, & Nakamura S. (2022). Structural basis for the toxin-coregulated pilus–dependent secretion of Vibrio cholerae colonization factor. Science Advances, 8(41), eabo3013.

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Radiolabeling and Annealing Oligonucleotides

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All oligonucleotides were purified via PAGE and reverse-phase chromatography (Sep-Pak classic C-18 cartridges). The 21-mer primer used for the single-turnover assays was 5′-[³²P]-labeled by incubating it with [γ−³²P]-ATP and OptiKinase (USB) for 3 hr at 37 °C. The reaction was terminated by heating at 95 °C for 2 min to denature Optikinase. The radiolabeled primer was purified from any unreacted [γ−³²P]-ATP using a Bio-spin 6 column (Bio-Rad). The primer was then annealed to a DNA template (Table 1) in a 1:1.35 molar ratio by first heating the reaction mixture to 95 °C for 5 min and then slowly cooling the mixture to room temperature overnight.

Tokarsky E.J., Wallenmeyer P.C., Phi K.K, & Suo Z. (2016). Significant impact of divalent metal ions on the fidelity, sugar selectivity, and drug incorporation efficiency of human PrimPol. DNA repair, 49, 51-59.

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Characterization of α7 and α6 Proteins

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The purified α7 and α6 proteins (420 μM and 510 μM monomer, respectively) were buffer-exchanged into 10 mM ammonium acetate, pH 6.8, and 150 mM ammonium acetate, pH 7.5, respectively, by passing the proteins through a Bio-Spin 6 column (Bio-Rad). The buffer-exchanged α7 (28 μM monomer) and α6 (5, 10, and 20 μM monomer) proteins were immediately analyzed by nanoflow electrospray ionization MS using gold-coated glass capillaries made in house (approximately 2–5 μL sample loaded per analysis). Buffer-exchanged α7 (2 μM tetradecamer) and α6 (0.5, 1, 2, 4, and 8 μM monomer) at pH 7.5 were mixed, incubated at 20 °C for 1 h, and analyzed by nanoflow electrospray ionization MS. Spectra were recorded on a SYNAPT G2-Si HDMS mass spectrometer (Waters, Manchester, UK) in positive ionization mode at 1.33 kV with a 150 V sampling cone voltage and source offset voltage, 0 V trap and transfer collision energy, and 5 mL/min trap gas flow. The spectra were calibrated using 1 mg/mL cesium iodide and analyzed using Mass Lynx software (Waters).

Ishii K., Noda M., Yagi H., Thammaporn R., Seetaha S., Satoh T., Kato K, & Uchiyama S. (2015). Disassembly of the self-assembled, double-ring structure of proteasome α7 homo-tetradecamer by α6. Scientific Reports, 5, 18167.

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Probing Kai and SasA Protein Interactions

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The purified TeKaiC_DD (20 µM) was titrated with either KaiB proteins (TeKaiB_10–108 and TeKaiB_10–108/DD) or SasA proteins (TeSasA_WT and TeSasA_N). The mixed protein solutions were incubated at 37 °C for 6 h, and were subsequently buffer-exchanged into 150 mM ammonium acetate, pH 6.8, using a Bio-Spin 6 column (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. In analyses of the Kai proteins from Synechococcus elongatus PCC 7942, the purified SyKaiC_DT (20 µM) was mixed with SyKaiB or SyKaiB_DD (30 µM), followed by incubation at 30 °C for 6 h. Approximately 2–5 µL of the buffer-exchanged protein solutions were immediately analyzed by nanoflow electrospray ionization MS using gold-coated glass capillaries prepared inhouse. Spectra were recorded on a SYNAPT G2-Si HDMS (Waters, Wilmslow, UK) in the positive ionization mode at 1.33 kV with a 150 V sampling cone voltage and source offset voltage, 0-V trap and transfer collision energy, and 5-mL/min trap gas flow. The spectra were calibrated using 1 mg/mL cesium iodide and analyzed using MassLynx software (Waters). Spectral measurements were performed at least in triplicate.

Murakami R., Yunoki Y., Ishii K., Terauchi K., Uchiyama S., Yagi H, & Kato K. (2019). Cooperative Binding of KaiB to the KaiC Hexamer Ensures Accurate Circadian Clock Oscillation in Cyanobacteria. International Journal of Molecular Sciences, 20(18), 4550.

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Prdx5 Redox Regulation by CoA

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Purified recombinant His-Prdx5 (0.5 µg) was incubated with a mixture of oxidised and reduced forms of CoA (CoASH and CoASSCoA, 1 mM final) in 20 mM Tris–HCl, pH 7.5 for 30 min at RT. The mixture was passed through a BioSpin 6 column (Bio-Rad) to remove excess CoA, and this preparation of Prdx5 was further used in activity assays. For Western blot analysis, NEM (10 mM final) was added to the samples for 10 min before mixing with SDS loading buffer (1× final) with or without DTT.
For SDS-PAGE analysis, 2 µg of His-Prdx5 was incubated with CoA and CoASSCoA as previously described, or with H₂O₂ (1 mM final) for 10 min or with buffer alone, and was mixed with reducing or non-reducing loading buffer, before loading on the gel.

Baković J., Yu B.Y., Silva D., Chew S.P., Kim S., Ahn S.H., Palmer L., Aloum L., Stanzani G., Malanchuk O., Duchen M.R., Singer M., Filonenko V., Lee T.H., Skehel M, & Gout I. (2019). A key metabolic integrator, coenzyme A, modulates the activity of peroxiredoxin 5 via covalent modification. Molecular and Cellular Biochemistry, 461(1), 91-102.

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FLAG-Tagged EDC4 Purification from HEK293T Cells

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FLAG-tagged EDC4 in pCMVSport6 was transfected into HEK293T cells using 10 µg DNA per T25 flask of cells. 10 flasks were used for each purification. 24 hours post-transfection, cells were harvested and lysed using tris-buffered saline (TBS) with 1% Triton X-100 (TBS-T), Roche Complete protease inhibitor cocktail tablets, and a phosphatase inhibitor tablet PhosSTOP (Sigma). 400 µL lysis buffer was used per pellet. Cells were lysed on ice for 20 min followed by centrifugation at 18000 × g at 4°C, 15 min. Lysate samples were saved for analysis of loading. A 50 µL aliquot of anti-FLAG agarose beads (clone M2, Sigma) was washed with 1 mL TBS-T, collected by centrifugation for 1 min at 1000 × g, then suspended in the cell lysate supernatant. Bead suspensions were rotated at 4°C for 2 hours, then washed 3 times with TBS-T, followed by extensive TBS washes. Elution was in 50 µL of 3× FLAG peptide (Sigma), at a final concentration of 1 mg/mL in TBS at 4°C for 1 hour. Beads were removed by centrifugation, excess FLAG peptide was removed using a Bio-Spin 6 column (BioRad), previously equilibrated in TBS. The protein concentration of FLAG-EDC4 eluted from the spin column was determined by measuring the absorbance at 280 nm.

D’Lima N.G., Ma J., Winkler L., Chu Q., Loh K.H., Corpuz E.O., Budnik B.A., Lykke-Andersen J., Saghatelian A, & Slavoff S.A. (2016). A human microprotein that interacts with the mRNA decapping complex. Nature chemical biology, 13(2), 174-180.

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Quantification of APAP Protein Adducts

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APAP protein adducts were measured as described in detail previously (Akakpo et al., 2018 (link)). Briefly, to remove low molecular weight metabolites that could interfere with detection of APAP protein adducts, liver homogenates were filtered through a Bio-Spin 6 column from Bio-Rad (Hercules, CA) (McGill et al., 2012b (link)). Then, 8 U/ml Streptomyces griseus solution was mixed with the filtrate containing proteins with APAP bound to cysteine residues in a 1:1 ratio. The mixture was digested for 15 hours at 50°C to release the APAP-Cys adducts from the cellular proteins. Finally, APAP-Cys residues derived from proteins were filtered and analyzed by high pressure liquid chromatography (HPLC) with a Coularray electrochemical detector from ESA Biosciences (Chelmsford, MA).

Akakpo J.Y., Ramachandran A., Orhan H., Curry S.C., Rumack B.H, & Jaeschke H. (2020). 4-Methylpyrazole Protects Against Acetaminophen-induced Acute Kidney Injury. Toxicology and applied pharmacology, 409, 115317.

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Doubly Nicked DNA Duplex Reconstitution

Cited 1 time

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A doubly nicked 30 bp DNA duplex was reconstituted using two asymmetric synthetic oligonucleotides obtained from Sigma-Aldrich. Sequences for the single strand 17 bp (5′-CGCGCATCGTCATCCTC-3′) and the single strand 13 bp (5′-GAGGATGACGATG-3′) were chosen as described in ref. ^{11 (link)}. Briefly, the nucleic acids were dissolved in DNAse-free water at 1 mM concentration. To assemble the double-stranded DNA, each oligo was mixed at 1:1 molar ratio, annealed by incubating at 95 °C for 2 min and then decreasing the temperature by 1 °C every 1 min until reaching 20 °C. The annealed doubly nicked DNA duplex was then buffer-exchanged in Hepes 20 mM pH 7.5 with a BioSpin 6 column (BioRad).

Vanden Broeck A., Lotz C., Drillien R., Haas L., Bedez C, & Lamour V. (2021). Structural basis for allosteric regulation of Human Topoisomerase IIα. Nature Communications, 12, 2962.

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Conjugation of Cy3-labeled ssDNA to ProG

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ProG (ab49807, Abcam. Recombinant ProG with 6-His tag for purification). Single-stranded DNA (ssDNA) was purchased from Integrated DNA technologies, Inc. The sequence and modifications are shown below: 5-/5Cy3/GGC CCG CAG CGA CCA CCC/3ThioMC3-D/ -3.

Add 5 μL × [50 mM TCEP (Tris(2-carboxyethyl)phosphine hydrochloride, reduction reagent, catalog#: 20490, Thermo Scientific) + 50 mM EDTA] (in PBS, PH7.2~7.4) into 20 μL × 1 mM thiol modified DNA in PBS. React for 30 mins at room temperature. Purpose: To deprotect thiol group by cleaving ThioMC3-D and make thiol group available for thiol-maleimide reaction.

Purify the DNA using Bio-spin 6 column (732–6200, Bio-rad. Buffer exchanged by PBS). Immediately add 1.5 μL × 23 mM sulfo-SMCC (22122, Thermo Scientific, pre-dissolved in pure water) into the DNA solution. React for 1 min.

Add the DNA solution into 10 μL × 5 mg/ml ProG (Buffer exchanged by DPBS). React overnight in 4 °C.

Purify the product using Dynabead (10103D, life technologies) through his-tag purification. Buffer exchange the final reagent with DPBS using Bio-spin 6 column.

Typical reading of final product: 100 μL × 19 μM DNA and 400 μg/ml (15 μM) ProG.

Wang X., Rahil Z., Li I.T., Chowdhury F., Leckband D.E., Chemla Y.R, & Ha T. (2016). Constructing modular and universal single molecule tension sensor using protein G to study mechano-sensitive receptors. Scientific Reports, 6, 21584.

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Characterization of Emp46p and Emp47p complexes

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The purified Emp46p^CC (or its E303A mutant) and Emp47p^CC proteins (100 μM) were buffer-exchanged into 200 mM ammonium acetate at pH 7.4 by passing through a Bio-Spin 6 column (Bio-Rad). The samples (43 μM) were immediately analyzed by nanoflow electrospray using in-house made gold-coated glass capillaries (approximately 2 μL sample loaded per analysis). The mixtures of Emp47p^CC (33 μM) and 17, 33, and 67 μM Emp46p^CC were incubated at 25°C for 30 min, and analyzed by electron spray ionization MS after the buffer exchange. Spectra were recorded on a SYNAPT G2-Si HDMS mass spectrometer (Waters, Manchester, UK) in positive ionization mode at 1.63 kV with 150 V sampling cone voltage and source offset voltage, 0 V trap and transfer collision energy, and 5 mL/min trap gas flow. The spectra were calibrated using 1 mg/mL cesium iodide and analyzed by Mass Lynx software (Waters).

Ishii K., Enda H., Noda M., Kajino M., Kim A., Kurimoto E., Sato K., Nakano A., Kobayashi Y., Yagi H., Uchiyama S, & Kato K. (2015). pH-Dependent Assembly and Segregation of the Coiled-Coil Segments of Yeast Putative Cargo Receptors Emp46p and Emp47p. PLoS ONE, 10(10), e0140287.

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