Biospin columns

Manufactured by Bio-Rad

Sourced in United States, United Kingdom

The Biospin columns are a versatile laboratory equipment used for the separation and purification of biomolecules, such as proteins, nucleic acids, and other macromolecules. These columns utilize a packed bed of matrix material to facilitate the efficient separation and isolation of the desired target from complex mixtures. The core function of the Biospin columns is to provide a reliable and reproducible method for the purification and enrichment of specific biomolecules, enabling researchers to obtain high-purity samples for further analysis and applications.

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Lab products found in correlation

γ 32p atp, by PerkinElmer (5 mentions) T4 polynucleotide kinase, by New England Biolabs (3 mentions) Dntps, by New England Biolabs (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) Quant it ribogreen rna assay kit, by Thermo Fisher Scientific (2 mentions) Pcr purification column, by Qiagen (2 mentions) Ampliscribe t7 flash transcription kit, by Illumina (2 mentions) Restriction endonuclease, by New England Biolabs (2 mentions) Protein g plus agarose, by Santa Cruz Biotechnology (2 mentions) Cnbr activated sepharose 4b beads, by Merck Group (2 mentions) J2 mc, by Beckman Coulter (1 mentions) Pbad topo ta expression kit, by Thermo Fisher Scientific (1 mentions) Fplc column, by GE Healthcare (1 mentions) Ptp1b, by Abcam (1 mentions) E coli uracil dna glycosylase udg, by New England Biolabs (1 mentions) Human pol β, by Bio-Techne (1 mentions) Acrylamide bis acrylamide solution, by Bio-Rad (1 mentions) T4 polynucleotide kinase t4 pnk, by New England Biolabs (1 mentions) Unlabeled dntps, by New England Biolabs (1 mentions) Streptavidin agarose bead, by Thermo Fisher Scientific (1 mentions)

24 protocols using biospin columns

Purification of His- and GST-tagged Proteins

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IPTG-induced BL21 cells (200 mL) were sonicated, and the cell lysate was centrifuged at 9000 × g (Beckman Coulter J2-MC, USA) at 4°C for 30 min. The supernatant was incubated in 500 μL of Ni-NTA agarose (Profinity IMAC NI-charged Resin, 1560131) at 4°C for 2 h. The agarose was washed five times with buffer containing 20 mM Tris (pH 8.0), 10 mM imidazole, 500 mM NaCl, and 10% glycerol, followed by a second wash with buffer containing 20 mM Tris (pH 8.0) and 100 mM NaCl. To elute the 6xHis-tagged proteins, 1 mL elution buffer containing 300 mM imidazole, 100 mM NaCl, and 20 mM Tris (pH 8.0) was used.
For purification of the GST-tagged protein, 500 μL of glutathione resin (Omics Bio, CL00206) was washed with PBS buffer. The resin was then added to the sample with 1% NP-40 and incubated at 4°C for 2 h. The mixture was transferred to bio-spin columns (Bio-Rad), washed five times with PBS buffer, followed by elution with 500 μL of elution buffer containing 20 mM glutathione, 150 mM NaCl, and 175 mM Tris buffer (pH 8.0). The amount of protein was quantified using the Bradford assay (Bradford, 1976 (link)).

Silamparasan D., Chang I.F, & Jinn T.L. (2023). Calcium-dependent protein kinase CDPK16 phosphorylates serine-856 of glutamate receptor-like GLR3.6 protein leading to salt-responsive root growth in Arabidopsis#. Frontiers in Plant Science, 14, 1093472.

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Protein Expression and Purification Protocol

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T4 polynucleotide kinase and dNTPs were purchased from New England Biolabs (Ipswich, MA). [γ−³²P]ATP (specific activity 3 × 10³ Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA). Bio-spin columns were purchased from Bio-Rad (Hercules, CA). A protease inhibitor cocktail was obtained from Roche Applied Science (Indianapolis, IN). The pBAD-TOPO TA expression kit was from Invitrogen (Carlsbad, CA) and the QuickChange mutagenesis kit was from Stratagene (La Jolla, CA). FPLC columns were purchased from GE Healthcare (Uppsala, Sweden).

Song I., Kim E.J., Kim I.H., Park E.M., Lee K.E., Shin J.H., Guengerich F.P, & Choi J.Y. (2014). Biochemical Characterization of Eight Genetic Variants of Human DNA Polymerase κ Involved in Error-free Bypass Across Bulky N2-Guanyl DNA Adducts. Chemical research in toxicology, 27(5), 919-930.

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Affinity Purification of HA-tagged Proteins

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Five 15-cm dishes of 50% confluent HA-STREP empty, HA-STREP-GFP and HA-STREP-SIRT2 Flp-In T-Rex 293 cell lines were induced with 1 µg ml⁻¹ doxycycline for 24 h prior to harvesting. Cells were harvested by scraping with cold PBS and lysed into cytoplasmic and chromatin fractions (Aygun et al., 2008 (link)). Fractions were applied to dust-free Biospin columns (BioRad) loaded with StrepTactin Sepharose (IBA) at 4°C. StrepTactin Sepharose was washed twice with the fractionation buffers containing 0.1% Triton X-100, followed by two washing steps without Triton X-100. Bound proteins were eluted with freshly prepared 2.5 mM biotin in the fractionation buffers without Triton X-100. The biotin eluate were subjected to co-immunoprecipitation with anti-HA magnetic beads (Pierce, Thermo Scientific) during a 2-h rotation at 4°C. Beads were washed six times with TBS with inhibitors. 80% of the beads were stored in 100 mM ammonium bicarbonate (ABC) and analysed by mass spectrometry following on-bead digestion, while the remaining 20% was eluted twice with glycine pH 2, neutralized with 1 M Tris-HCl pH 9.2 and used for western blotting.

Kaufmann T., Kukolj E., Brachner A., Beltzung E., Bruno M., Kostrhon S., Opravil S., Hudecz O., Mechtler K., Warren G, & Slade D. (2016). SIRT2 regulates nuclear envelope reassembly through ANKLE2 deacetylation. Journal of Cell Science, 129(24), 4607-4621.

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Optimizing SPOP Protein MS Analysis

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Prior to MS analysis, SPOP was diluted to 30 μM with 150 mM ammonium acetate (pH 7.5) and 5 mM DTT and buffer‐exchanged into the same buffer, using Bio‐Spin columns (Bio‐Rad). Typically, aliquots of 2 μl of protein sample were electrosprayed from gold‐coated borosilicate capillaries prepared in‐house, as previously described (Kirshenbaum et al, 2010). Nanoflow electrospray ionization MS experiments were then conducted under non‐denaturing conditions on a QToF Q‐Star Elite instrument (MDS Sciex, Canada), modified for improved transmission of large non‐covalent complexes. Assays were performed in positive ion mode. Conditions were optimized to enable the ionization and removal of adducts, without disrupting the non‐covalent interactions of the proteins tested. The following experimental parameters were used: ionspray voltage, 1,250 V; declustering potential, 180 V; focusing potential, 250 V; second declustering potential, 5 V; and collision energy, 60 V. Spectra are shown with minimal smoothing and without background subtraction.

Marzahn M.R., Marada S., Lee J., Nourse A., Kenrick S., Zhao H., Ben‐Nissan G., Kolaitis R., Peters J.L., Pounds S., Errington W.J., Privé G.G., Taylor J.P., Sharon M., Schuck P., Ogden S.K, & Mittag T. (2016). Higher‐order oligomerization promotes localization of SPOP to liquid nuclear speckles. The EMBO Journal, 35(12), 1254-1275.

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In vitro RNA Characterization Assay

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dsDNA from the brightest bacterial colonies was PCR amplified from the purified plasmids. Truncation, deletion and point mutation mutants were generated from dsDNAs that were PCR amplified from the appropriate ssDNA templates. Where indicated, dsDNA, and thus the encoded RNA, also contained the tRNA scaffold sequence. PCR products were then purified with PCR purification columns (Qiagen) and in vitro transcribed utilizing an AmpliScribe T7-Flash Transcription Kit (Epicenter). RNA was purified using Bio-Spin columns (Bio-Rad), and quantified using both absorbance values and the Quant-iT RiboGreen RNA Assay Kit (Life Technologies).
Absorption, excitation and emission spectra were measured for solutions using “excess RNA” conditions and limiting amount of fluorophore to ensure that no free fluorophore contributes to the absorbance or fluorescence signal^{11 (link)}. The RNA concentration was 20 μM (for the fluorescence measurements) and 50 μM (for the absorbance measurements) while the DFHO concentration was 2 μM and 5 μM respectively.

Song W., Filonov G.S., Kim H., Hirsch M., Li X., Moon J.D, & Jaffrey S.R. (2017). Imaging RNA polymerase III transcription using a photostable RNA-fluorophore complex. Nature chemical biology, 13(11), 1187-1194.

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Oxidative Modification and Labeling of PTP1B

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PTP1B, human recombinant, was purchased from Abcam at 1 mg/mL. Protein was desalted on biospin columns (BioRad) and concentration adjusted to 10 μM. Samples were either left untreated, exposed to 50 μM H₂O₂, or combination of 50 μM H₂O₂ and H₂S for 15 min at 37 °C. Samples were then desalted and treated with or without 1 mM NBF-Cl in HEPES buffer supplemented with 2 % SDS for 1 h at 37 °C. Following a desalting step on biospin column, the same samples were treated with 20 μM DAz-2:Cy-5 Click Mix for 30 min at at 37 °C. Unreacted reagent was removed by desalting on biospin columns and samples mixed with Laemmli buffer supplemented with 10% β-mercaptoethanol.

Zivanovic J., Kouroussis E., Kohl J.B., Adhikari B., Bursac B., Schott-Roux S., Petrovic D., Miljkovic J.L., Thomas-Lopez D., Jung Y., Miler M., Mitchell S., Milosevic V., Gomes J.E., Benhar M., Gonzalez-Zorn B., Ivanovic-Burmazovic I., Torregrossa R., Mitchell J.R., Whiteman M., Schwarz G., Snyder S.H., Paul B.D., Carroll K.S, & Filipovic M.R. (2019). SELECTIVE PERSULFIDE DETECTION REVEALS EVOLUTIONARILY CONSERVED ANTI-AGING EFFECTS OF S-SULFHYDRATION. Cell metabolism, 30(6), 1152-1170.e13.

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Recombinant Human Polymerases for Gel Electrophoresis

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Full-length recombinant human polymerase κ (hPol κ) used for gel electrophoresis experiments was purchased from Enzymax (Lexington, KY). Human Pol β was obtained from Trevigen (Gaithersburg, MD). Recombinant human DNA polymerases hPol η (amino acids 1–437), hPol ι (amino acids 1–420), and hPol κ (amino acids 19–526) (active core enzymes) were expressed and purified according to previously published procedures.^{44 (link)–46 (link)} T4 polynucleotide kinase (T4-PNK) and E. coli uracil DNA glycosylase (UDG) were purchased from New England Biolabs (Beverly, MA). [γ-³²P]ATP was purchased from Perkin-Elmer Life Sciences (Boston, MA). Acrylamide/bis-acrylamide solution (40% 19:1, w/w) and Bio-spin columns were obtained from Bio-Rad laboratories (Hercules, CA). All other chemicals were purchased either from Sigma-Aldrich or Fisher Scientific.

Kotapati S., Wickramaratne S., Esades A., Boldry E.J., Dorr D.Q., Pence M.G., Guengerich F.P, & Tretyakova N.Y. (2015). Polymerase Bypass of N6-Deoxyadenosine Adducts Derived from Epoxide Metabolites of 1,3-Butadiene. Chemical research in toxicology, 28(7), 1496-1507.

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Radioactive DNA Labeling Protocol

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T4 polynucleotide kinase, restriction endonucleases and unlabeled dNTPs were purchased from New England Biolabs (Beverly, MA). [γ-³²P]ATP (specific activity 3×10³ Ci mmol⁻¹) was obtained from PerkinElmer Life Sciences (Boston, MA). Bio-spin columns were obtained from Bio-Rad (Hercules, CA). Other reagents were of the highest quality commercially available.

Xue Q., Zhong M., Liu B., Tang Y., Wei Z., Guengerich F.P, & Zhang H. (2015). Kinetic analysis of bypass of 7,8-dihydro-8-oxo-2′-deoxyguanosine by the catalytic core of yeast DNA polymerase η. Biochimie, 121, 161-169.

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Streptavidin-Agarose Bead Purification

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100 μl of streptavidin-agarose beads (ThermoFisher Scientific) were washed with PBS in biospin columns and added to the protein samples, followed by incubation with rotation for 1.5 h at room temperature. The beads were pelleted by centrifugation at 1400 g for 2 minutes, transferred into biospin columns (Bio-Rad) and washed 3 times with 1% SDS followed by three washes with 6 M urea.

Filip R., Desrochers G.F., Lefebvre D.M., Reed A., Singaravelu R., Cravatt B.F, & Pezacki J.P. (2021). Profiling of microRNA targets using activity-based protein profiling: linking enzyme activity to microRNA-185 function. Cell chemical biology, 28(2), 202-212.e6.

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Immunoprecipitation and Mass Spectrometry of Flot2

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All immunoprecipitations were performed from postconfluent 3T3-L1 cells. Lysis was done in RIPA buffer (50mMTris [pH 7.4], 150mMNaCl, 1% Triton X-100, with 60 mM octyl glucopyranoside), based on [15] (link). Briefly, cell lysate was incubated overnight at 4 °C on an overhead rotator with 5 μg of antibody (Santa Cruz Biotechnology FLOT2 SC-25507 or rabbit IgG control SC-2027). Next, 60 μl of Protein G-PLUS Agarose (Santa Cruz) (pre-washed twice with RIPA buffer) was incubated with lysate for 2 h at 4 °C on overhead rotator. Agarose beads were washed 5 times (RIPA, 1000 g spin for 2 min between washes), and proteins were eluted in supernatant for western blots in 100 μl of 2× Laemmli buffer (boiled for 10 min before a brief full speed spin to pellet the remaining agarose). For mass spectrometry, agarose beads were transferred to Bio-Spin columns (Bio Rad, #732-6204) for 5 washes with 50 mM ammonium bicarbonate buffer. Proteins were digested overnight at 37 °C with 0.5 μg trypsin in 50 mM ammonium bicarbonate buffer, and peptides in supernatant were collected and frozen.

Grandl G., Müller S., Moest H., Moser C., Wollscheid B, & Wolfrum C. (2016). Depot specific differences in the adipogenic potential of precursors are mediated by collagenous extracellular matrix and Flotillin 2 dependent signaling. Molecular Metabolism, 5(10), 937-947.

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