35S-sodium sulfate (1,500 Ci/mmol) was from PerkinElmer. Sulfate-free Dulbecco’s modified Eagle’s medium (DMEM) (AS31600 cat no. 074-91083P) was from Gibco. Superose 6 10/300, Superdex Peptide 10/300, PD-10 columns, Sephadex G-25, DEAE-Sephacel were from Cytiva. DS-epi1 was detected by Western blot with 1 μg/mL of an immunopurified anti-DS-epi1 rabbit polyclonal antibody obtained by immunization with the peptide antigen (KWSKYKHDLAAS, corresponding to amino acids 509 to 520 of the human/murine sequence; Innovagen, Sweden) (Maccarana et al. 2006 (link)). The Biorad stain-free gels were used, and both loading control, i.e. the blotted bands, and the chemioluminiscent signal were visualized with the ChemiDoc Imaging System (Biorad). Cell number was evaluated by DNA staining with crystal violet. Viability was assessed with PrestoBlue™ Cell Viability Reagent (Invitrogen).
Superdex peptide 10 300
Manufactured by Cytiva
Sourced in Sweden
Superdex Peptide 10/300 is a size exclusion chromatography column designed for the purification and analysis of peptides and small proteins. The column is packed with a bead-formed, highly cross-linked agarose-based matrix that provides high resolution and fast separation. Superdex Peptide 10/300 can be used with a variety of common buffer systems and is suitable for use with analytical and preparative applications.
Automatically generated - may contain errors
Lab products found in correlation
Pd 10 column, by Cytiva (2 mentions)
Sulfate free dulbecco s modified eagle s medium dmem as31600 cat no 074, by Thermo Fisher Scientific (2 mentions)
Deae sephacel, by Cytiva (2 mentions)
Sephadex g 25, by Cytiva (2 mentions)
35s sodium sulfate, by PerkinElmer (2 mentions)
Superose 6 10 300, by Cytiva (2 mentions)
Prestoblue cell viability reagent, by Thermo Fisher Scientific (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Ebselen, by Merck Group (1 mentions)
Superdex 75 increase 10 300, by Cytiva (1 mentions)
3 protocols using superdex peptide 10 300
1
Quantitative Assessment of DS-epi1 Levels
2
Ebselen Sulfate Metabolism Assay
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Ebselen was purchased from Sigma and 50 mM stock solution was prepared in DMSO. 35S-sodium sulfate (1500 Ci/mmol) was from PerkinElmer. Sulfate-free Dulbecco’s modified Eagle’s medium (DMEM) (AS31600 cat no. 074-91083P) was from Gibco. Superose 6 10/300, Superdex Peptide 10/300, PD-10 columns, Sephadex G-25, DEAE-Sephacel were from Cytiva.
3
Quantitative Analysis of SUMO-SIM Binding
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SUMO and SIM were buffer-exchanged
using a Superdex75Increase 10/300 or Superdex Peptide 10/300 gel filtration
column (Cytiva Life Sciences) into 20 mM HEPES, 150 mM KCl, 1 mM MgCl2, 1 mM DTT, 1 mM EGTA (pH 7.0 or 8.0) or 20 mM MES, 150 mM
KCl, 1 mM MgCl2, 1 mM DTT, and 1 mM EGTA (pH 6.5). ITC
experiments were performed using a Microcal ITC200 isothermal calorimeter
using 50–100 μM SIM peptide in the cell and 750–1.2
mM SUMO in the syringe. Baseline corrections and integrated heats
were performed using NITPIC.68 (link) Affinity
was determined using a 1:1 binding model in SEDPHAT.69 (link) Reported error is the standard deviation from the Monte
Carlo simulation based on the experimental noise.
using a Superdex75Increase 10/300 or Superdex Peptide 10/300 gel filtration
column (Cytiva Life Sciences) into 20 mM HEPES, 150 mM KCl, 1 mM MgCl2, 1 mM DTT, 1 mM EGTA (pH 7.0 or 8.0) or 20 mM MES, 150 mM
KCl, 1 mM MgCl2, 1 mM DTT, and 1 mM EGTA (pH 6.5). ITC
experiments were performed using a Microcal ITC200 isothermal calorimeter
using 50–100 μM SIM peptide in the cell and 750–1.2
mM SUMO in the syringe. Baseline corrections and integrated heats
were performed using NITPIC.68 (link) Affinity
was determined using a 1:1 binding model in SEDPHAT.69 (link) Reported error is the standard deviation from the Monte
Carlo simulation based on the experimental noise.
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