Desthiobiotin

Manufactured by Merck Group

Sourced in United States

Desthiobiotin is a chemical compound used as a lab equipment product. It is a structural analog of biotin, a vitamin commonly used in biochemical and molecular biology applications. Desthiobiotin can be used for various research purposes, including affinity purification and protein labeling.

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Lab products found in correlation

Autoinduction medium, by Formedium (5 mentions) Hispur cobalt resin, by Thermo Fisher Scientific (4 mentions) Protease inhibitor cocktail, by Roche (3 mentions) Streptactin column, by GE Healthcare (3 mentions) Streptrap column, by GE Healthcare (3 mentions) Streptrap hp, by GE Healthcare (3 mentions) Strep tactin superflow plus resin, by Qiagen (2 mentions) Flag peptide, by GenScript (2 mentions) Flag affinity beads, by Merck Group (2 mentions) Strep affinity beads, by Merck Group (2 mentions) Streptactin sepharose beads, by Cytiva (2 mentions) Sm2 bio beads, by Bio-Rad (2 mentions) Pierce protease inhibitor edta free tablet, by Thermo Fisher Scientific (2 mentions) Superose 6 16 70 or increase 10 300 column, by GE Healthcare (2 mentions) Hitrap heparin hp column, by GE Healthcare (2 mentions) Benzonase, by Merck Group (2 mentions) Neomycin, by Thermo Fisher Scientific (1 mentions) Freestyle 293 expression system, by Thermo Fisher Scientific (1 mentions) Gas phase sequencer model 492, by Thermo Fisher Scientific (1 mentions) Model 140c hplc system, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

D-desthiobiotin (33 citations)

46 protocols using desthiobiotin

Production and Purification of Recombinant C1r

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Recombinant p.W435R variant of C1r was produced in the FreeStyle 293 Expression System (Thermo Fisher), using a pcDNA3.1/Neo(+) plasmid encoding human C1r with a C-terminal Strep-tag. 293-F cells grown in FreeStyle 293 medium were transfected with this plasmid using 293 fectin and stable transfectants were selected with 400 μg/ml neomycin (Thermo Fisher). Recombinant C1r was purified from the culture supernatant by chromatography on StrepTactin Sepharose High performance (Sigma-GE28-9355-99), as recommended by the manufacturer. Briefly, the cell culture supernatant was concentrated 6-fold, dialyzed against 100 mM Tris, 150 mM NaCl, 1 mM EDTA, pH 8.0, and applied to a 1-ml column equilibrated in the same buffer. Elution was carried out using 2.5 mM desthiobiotin (Sigma-Aldrich) in the same buffer. The eluted protein was dialyzed in the same buffer and concentrated to 0.3–0.45 mg/ml. N-terminal sequence determination of the major band observed after SDS-PAGE analysis under non-reducing conditions was performed using an Applied Biosystems gas-phase sequencer model 492 coupled online with an Applied Biosystems Model 140C HPLC system.

Gröbner R., Kapferer-Seebacher I., Amberger A., Redolfi R., Dalonneau F., Björck E., Milnes D., Bally I., Rossi V., Thielens N., Stoiber H., Gaboriaud C, & Zschocke J. (2019). C1R Mutations Trigger Constitutive Complement 1 Activation in Periodontal Ehlers-Danlos Syndrome. Frontiers in Immunology, 10, 2537.

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Tandem Affinity Purification Mass Spectrometry

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TAP-MS was performed as previously described (Pu et al., 2015 (link)). In brief, HeLa or H4 cells stably expressing proteins tagged with N-terminal OSF or C-terminal FOS were extracted with 1% Triton X-100, 50 mM Tris-HCl, pH 7.4, 300 mM NaCl, and 5 mM EDTA, supplemented with proteinase inhibitor cocktail (Roche). After clearing by centrifugation for 15 min at 17,000 g, proteins were sequentially purified on Strep-Tactin (IBA) and FLAG antibody–coated beads (Sigma-Aldrich). Beads were washed with 0.2% Triton X-100, 50 mM Tris-HCl, pH 7.4, 300 mM NaCl, and 5 mM EDTA, and bound proteins were eluted with 2.5 mM desthiobiotin and 150 ng/ml of 3× FLAG peptide (Sigma-Aldrich), respectively. Proteins were precipitated with 10% TCA, washed with acetone, air-dried, and analyzed by liquid chromatography (LC)/MS at the Taplin MS facility (Harvard Medical School, Boston, MA). The results of MS analysis for OSF-BLOS2 are shown in Table S1 from Pu et al. (2015) (link), and those for OSF-lyspersin, lyspersin-FOS, and OSF-Pallidin are shown in in Tables S1, S2, and S3 of the current study.

Pu J., Keren-Kaplan T, & Bonifacino J.S. (2017). A Ragulator–BORC interaction controls lysosome positioning in response to amino acid availability. The Journal of Cell Biology, 216(12), 4183-4197.

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Antibiotic and Induction Compound Preparation

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Carbenicillin, kanamycin, chloramphenicol, and β-D-1-thiogalactopyranoside (IPTG) were purchased from Gold Bio (St. Louis, MO, USA). Desthiobiotin, NaAD were acquired from Sigma (St. Louis, MO, USA). All other chemicals used were reagent grade or better.

Turmo A., Hu J, & Hausinger R.P. (2022). Characterization of the nickel-inserting cyclometallase LarC from Moorella thermoacetica and identification of a cytidinylylated reaction intermediate. Metallomics: Integrated Biometal Science, 14(3), mfac014.

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Recombinant Viral Protein Expression

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Protein expression and purification were performed as described previously (Chang et al., 2019 (link)). The recombinant VSV-G, CoV-S, and CoV-2 S and the variant proteins in our study are soluble forms of the proteins with the transmembrane domains removed. Briefly, HEK-293 GnTI cells that had been transfected with plasmids expressing 2 × Strep-tagged VSV-G (amino acids 1–464), SARS-CoV S (amino acids 1–1190), or SARS-CoV-2 S and its variants (amino acids 1–1208) were harvested in the lysis buffer (50 mM HEPES, pH 7.5, 1% Triton X-100, 150 mM NaCl, 1 mM MgCl₂, 10% glycerol, and 1 mM dithiothreitol [DTT]) supplemented with protease inhibitors (Roche). The clarified lysates were incubated with Strep-Tactin Superflow beads (IBA) for 3 h at 4°C. The beads were washed extensively with the washing buffer (50 mM HEPES, pH 7.5, 300 mM NaCl, 1 mM MgCl₂, and 1 mM DTT), and the Strep-fusion proteins were eluted with 10 mM desthiobiotin (Sigma) in the elution buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, and 1 mM DTT) and concentrated with Amicon Ultra Centrifugal Filters (Millipore).

Lu Y., Zhu Q., Fox D.M., Gao C., Stanley S.A, & Luo K. (2022). SARS-CoV-2 down-regulates ACE2 through lysosomal degradation. Molecular Biology of the Cell, 33(14), ar147.

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Purification of CAK Complexes

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CAK complexes were purified using Nickel affinity, Strep-Tactin affinity, and size exclusion chromatography. Cells were resuspended in lysis buffer (250 mM KCl, 40 mM Hepes-KOH pH 7.9, 5 mM MgCl₂, 5 mM β-mercaptoethanol, 10 mM imidazole, and 10% [vol/vol] glycerol supplemented with protease inhibitors and DNaseI) and homogenized by sonication. The lysate was cleared using a JA-20 rotor (18,000 rpm for 30 min at 4 °C) and incubated with Ni-NTA superflow resin (Qiagen) for 30 min. The beads were washed in purification buffer (250 mM KCl, 25 mM Hepes-KOH pH 7.9, 5 mM MgCl₂, 5 mM β-mercaptoethanol, and 10% [vol/vol] glycerol) supplemented with 25 mM imidazole and eluted in purification buffer with 300 mM imidazole. The eluate was incubated with Strep-Tactin superflow plus resin (Qiagen) for 30 min, washed with 10 bed volumes of purification buffer, and eluted with purification buffer supplemented with 10 mM desthiobiotin (Sigma-Aldrich). The eluted fractions were concentrated by ultrafiltration using 30-kDa molecular weight cutoff Centricon centrifugal filter units (EMD Millipore) and applied onto a Superdex 200 10/300 GL column (GE Healthcare) for size exclusion chromatography (SI Appendix, Fig. S1B). Purified fractions (SI Appendix, Fig. S1C) were pooled, concentrated to 2 mg/mL, and frozen in liquid N₂ for storage at −80 °C.

Greber B.J., Perez-Bertoldi J.M., Lim K., Iavarone A.T., Toso D.B, & Nogales E. (2020). The cryoelectron microscopy structure of the human CDK-activating kinase. Proceedings of the National Academy of Sciences of the United States of America, 117(37), 22849-22857.

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Purification of Prefusion RSVF Trimer

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Protein sequence of prefusion RSVF corresponds to the sc9-10 DS-Cav1 A149C Y458C S46G E92D S215P K465Q variant designed by Joyce and colleagues [42 (link)], which we refer to as RSVF DS2. RSVF DS2 was codon optimized for mammalian expression and cloned into the pHCMV-1 vector together with two C-terminal Strep-Tag II and one 8x His tag. Plasmids were transfected in HEK293-F cells and cultured in FreeStyle medium. Supernatants were harvested 1 week after transfection and purified via Ni-NTA affinity chromatography. Bound protein was eluted using buffer containing 10 mM Tris, 500 mM NaCl, and 300 mM Imidazole (pH 7.5), and eluate was further purified on a StrepTrap HP affinity column (GE Healthcare). Bound protein was eluted in 10 mM Tris, 150 mM NaCl and 20 mM desthiobiotin (pH 8) (Sigma) and size excluded in PBS (pH 7.4) on a Superdex 200 Increase 10/300 GL column (GE Healthcare) to obtain trimeric RSVF.

Sesterhenn F., Galloux M., Vollers S.S., Csepregi L., Yang C., Descamps D., Bonet J., Friedensohn S., Gainza P., Corthésy P., Chen M., Rosset S., Rameix-Welti M.A., Éléouët J.F., Reddy S.T., Graham B.S., Riffault S, & Correia B.E. (2019). Boosting subdominant neutralizing antibody responses with a computationally designed epitope-focused immunogen. PLoS Biology, 17(2), e3000164.

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Strep, Myc and FLAG Pulldown Assay

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For Strep, Myc and FLAG pulldown experiments proteins were immobilized using either Strep-Tactin Superflow Plus (QIAGEN), Anti-c-Myc Agarose (Thermo Fisher) or Anti-FLAG M2 affinity resin (Sigma). Proteins were added and incubated with the resin for 15 minutes at room temperature. Beads were washed three times with 15-20 bed volumes each. Bound proteins were eluted using either 2.5 mM desthiobiotin (Sigma), 500 μg/ml Myc peptide (GenScript) or 100 μg/ml 3X FLAG peptide (Sigma) and analyzed by SDS-PAGE.

Schreiber A., Collins B.C., Davis C., Enchev R.I., Sedra A., D’Antuono R., Aebersold R, & Peter M. (2021). Multilayered regulation of autophagy by the Atg1 kinase orchestrates spatial and temporal control of autophagosome formation. Molecular Cell, 81(24), 5066-5081.e10.

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Affinity Purification and Immunoprecipitation of VHL

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ACHN cells were infected with Lentiviral plasmids encoding SF-tagged (Strep II tag and Flag tag)-VHL to establish stable cell lines expressing SF-VHL proteins. Cells (~6 × 10⁷) were lysed in NETN buffer containing protease inhibitor cocktail (Roche). The cell lysates were centrifuged at 13,000 × g for 10 min at 4°C to remove debris and then incubated with Strep-Tactin Sepharose (IBA, Göttingen, Germany) for 4 h at 4°C. The complexes were washed three times with NETN buffer and then bound proteins were eluted with NETN buffer containing 2.5 mM desthiobiotin (Sigma-Aldrich). The eluents were incubated with anti-ZBRK1 antibody and Protein G Agarose for overnight at 4°C. After three washes, the immunocomplexes were analyzed by Western blot with the appropriate antibodies.

Chen K., Yu G., Gumireddy K., Li A., Yao W., Gao L., Chen S., Hao J., Wang J., Huang Q., Xu H, & Ye Z. (2015). ZBRK1, a novel tumor suppressor, activates VHL gene transcription through formation of a complex with VHL and p300 in renal cancer. Oncotarget, 6(9), 6959-6976.

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Expression and Purification of Cas1-Cas2/3

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E. coli BL21(DE3) cells were transformed with pCas1Cas23 and grown 2× 1 L LB-Miller media (10 g/L Tryptone, 10 g/L NaCl, 5 g/L yeast extract), supplemented with 50 μg/mL Spectinomycin, at 37°C and 200 rpm to an OD₆₀₀ of 0.45. Cultures were then cooled on ice for two hours, without agitation, before being induced with 0.2 mM IPTG. Cells were then grown for an additional 18 hours at 16°C, before being centrifuged at 5000 g for 10 minutes. Each cell pellet, originating from 1 L of culture, was resuspended in 20 mLs of Cas1-2/3 Lysis Buffer (50 mM HEPES pH 7.5, 500 mM KCl, 10% Glycerol) supplemented with 0.3x Halt™ Protease Inhibitor Cocktail (ThermoFisher). Cells were lysed via sonication, and lysate was clarified as above. StrepII-tagged Cas1-Cas2/3 was affinity purified on StrepTrap HP resin (GE Healthcare) and eluted with Cas1-2/3 Lysis Buffer containing 3 mM desthiobiotin (Sigma-Aldrich). Eluate was concentrated at 4°C (Corning Spin-X concentrators), before purification over a Superdex 200 size-exclusion column (Cytiva) equilibrated in 10 mM HEPES pH 7.5, 500 mM KCl, and 10% Glycerol.

Santiago-Frangos A., Buyukyoruk M., Wiegand T., Krishna P, & Wiedenheft B. (2021). Distribution and phasing of sequence motifs that facilitate CRISPR adaptation. Current biology : CB, 31(16), 3515-3524.e6.

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Purification of Dynamin1, EHD1, and mEGFP-LactC2

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cDNAs for human dynamin1 (Q05193) and human EHD1 (Q9H4M9) were cloned with N-terminal six-His and C-terminal StrepII tags in pET15B using polymerase chain reaction. mEGFP-LactC2 was a gift from S. Grinstein (Addgene plasmid 22852) and was cloned in the same vector. All proteins were expressed in BL21(DE3) grown in autoinduction medium (Formedium, Norfolk, U.K.) for 36–48 h at 18 °C. Cell pellets were stored frozen at −40 °C. For purification, frozen cell pellets were thawed in 20 mM HEPES (pH 7.4) buffer with 300 mM KCl and sonicated in an ice–water bath. Lysates were spun at 20000g, and the supernatant was incubated with HisPur Cobalt resin (Thermo Scientific) for 1 h at 4 °C. The resin was then poured into a PD-10 column, washed with 100 mL of the same buffer, and eluted with 20 mM HEPES (pH 7.4) buffer with 300 mM KCl and 150 mM imidazole. The elution was then applied to a 5 mL Streptactin column (GE Lifesciences) and washed with 20 mM HEPES (pH 7.4) buffer with 150 mM KCl. Bound protein was eluted in 20 mM HEPES (pH 7.4) buffer with 150 mM KCl and 2.5 mM desthiobiotin (Sigma). Purified proteins were spun at 100000g to remove aggregates before use in microscopic assays. All nucleotides were obtained from Jena Bioscience.

Kamerkar S.C., Roy K., Bhattacharyya S, & Pucadyil T.J. (2018). A Screen for Membrane Fission Catalysts Identifies the ATPase EHD1. Biochemistry, 58(1), 65-71.

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