BSA was labeled with tetramethylrhodamine-5-isothiocyanate at a ratio of 1:2 at pH 8.6. The labeled mixture was passed four times before measurements through a DEAE Sepharose (GE Healthcare) column to remove extra fluorescent probes and dialysis against PBS. Unlabeled goat anti-mouse IgG (Jackson ImmunoResearch) was conjugated to QDs-540-amine using the appropriate LYNX rapid conjugation kit (AbD Serotec). Human cTnT were over expressed in E. coli strain BL21(DE3) cells and purified according to our previous studies [30 (link)] and then labeled Alexa Fluor 488, with a ratio of 1:2 at pH 8.6, the labeled mixture was first passed a DEAE Sepharose (GE Healthcare) column to remove extra fluorescent probe and dialysis against PBS for four times before measurements.
Deae sepharose
Manufactured by GE Healthcare
Sourced in United States, United Kingdom, Sweden
DEAE-Sepharose is a chromatography resin used for the purification and separation of biomolecules, such as proteins, enzymes, and nucleic acids. It is an anion exchange material that can be used in a variety of applications, including protein purification, enzyme isolation, and DNA/RNA separation.
Automatically generated - may contain errors
Lab products found in correlation
Q sepharose, by GE Healthcare (3 mentions)
Superdex 200, by GE Healthcare (3 mentions)
Vivaspin concentrator, by GE Healthcare (2 mentions)
Pmal c2x, by New England Biolabs (2 mentions)
Atp agarose, by Merck Group (2 mentions)
Tris base, by Merck Group (2 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (2 mentions)
Acrylamide, by Merck Group (2 mentions)
Glycine, by Merck Group (2 mentions)
Cm sepharose, by GE Healthcare (2 mentions)
Pd 10 column, by GE Healthcare (2 mentions)
Sephadex g 25, by GE Healthcare (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Mouse monoclonal igg1, by Santa Cruz Biotechnology (1 mentions)
Amicon centrifugal filter, by Merck Group (1 mentions)
Hitrap q hp column, by GE Healthcare (1 mentions)
Superose 6 column, by GE Healthcare (1 mentions)
Rpmi 1640 media, by PanEco (1 mentions)
Ypd media, by Merck Group (1 mentions)
Sabouraud dextrose agar, by Merck Group (1 mentions)
35 protocols using deae sepharose
1
Protein Labeling and Purification Protocol
2
Immunoprecipitation of RAD52 and RPA
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U2OS cells (106 cells) were seeded in a 100 mm dish and grown overnight. The cells were treated with the indicated concentrations of mitoxantrone (MTX) for 24 hours. The cell lysate was prepared in 100 μl of lysis buffer (PBS with 0.5% NP40, 0.5% Triton X100, 1 mM DTT, protease and phosphatase inhibitors, and 10% glycerol). After mixing with 100 μl cold PBS, the lysate was incubated with 100 μl of DEAE sepharose (GE Life Sciences) for 2 hours at 4°C with rotation. An unbound fraction from DEAE sepharose (~200 μl) was diluted with 200 μl of PBS and half of the mixture was fractionated with Superdex200 (GE Life Sciences) using PBS as elution buffer. Each fraction was incubated with anti-RPA (p32) antibody conjugated with biotin (NeoMarkers, MS-691-B0) at 4°C overnight, then incubated with Streptavidin-magnetic beads (Promega #Z5481) for one hour at 4°C with rotation. After washing with PBS, the immunoprecipitated proteins were eluted with 1xSDS loading dye and separated by 10% SDS-PAGE. RAD52 and RPA (p32) were detected by western blotting with anti-RAD52 antibody (LS-C42570, LSBio) and anti-RPA2 antibody (A300-244A, Bethyl Laboratories), respectively.
3
Purification of Recombinant hPLSCR1 Protein
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hPLSCR1 cDNA clone was obtained from Origene, MD, USA, and was subsequently cloned into pET-28a (+) bacterial expression vector with an N-terminal His tag. This was transformed into E. coli BL-21 (DE3) and grown in LB media containing kanamycin (50 mg/l). Overexpression and purification were performed as described earlier [34 (link)]. Briefly, cells were lysed in buffer (20 mM Tris–HCl (pH – 7.4), 200 mM NaCl) by sonication, hPLSCR1 formed inclusion bodies (IB). N- lauroylsarcosine (N-LS) was used to recover native protein from IB followed by dialysis to remove N-LS and the proteins were purified to homogeneity using Ni2+-NTA chromatography. The eluted fractions from Ni2+-NTA chromatography were subjected to anion-exchange chromatography where DEAE sepharose (GE Healthcare, LC, UK) was used. The NaCl eluted protein fractions were again loaded in Ni2+-NTA resin and eluted. The purified protein was further concentrated by Amicon centrifugal filters (10 kDa cut-off) (Millipore, MA, USA) and visualized by silver staining or coomassie staining and confirmed by western blotting using anti-hPLSCR1 monoclonal antibody (Name: PLSCR1 antibody 1E9; catalog no - sc59645; specificity -human; origin - mouse monoclonal IgG1, Santa Cruz Biotechnology, TX, USA).
4
Preparation of Amyloid-Beta Peptides
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All chemical compounds used in intracellular and extracellular solutions were obtained from Sigma-Aldrich Sweden AB (Stockholm, Sweden). CCh was dissolved in milliQ water.
Met-Aβ residues 1–42 (referred to as Aβ herein) were produced as previously described38 . Briefly, Aβ was expressed in BL21*(DE3) pLysS Escherichia coli and purified with DEAE-Sepharose (GE Healthcare). To get rid of large aggregates, the peptides were passed through a 30,000 Da Vivaspin concentrator (GE Healthcare) at 4 °C. The filtrate was concentrated at 4 °C with a 5000 Da Vivaspin concentrator (GE Healthcare) until Aβ concentration was ~50 µM. The peptides were aliquoted in low-bind Eppendorf tubes (Axygen) and stored at −20 °C.
Met-Aβ residues 1–42 (referred to as Aβ herein) were produced as previously described38 . Briefly, Aβ was expressed in BL21*(DE3) pLysS Escherichia coli and purified with DEAE-Sepharose (GE Healthcare). To get rid of large aggregates, the peptides were passed through a 30,000 Da Vivaspin concentrator (GE Healthcare) at 4 °C. The filtrate was concentrated at 4 °C with a 5000 Da Vivaspin concentrator (GE Healthcare) until Aβ concentration was ~50 µM. The peptides were aliquoted in low-bind Eppendorf tubes (Axygen) and stored at −20 °C.
5
Cloning and Purification of P. vivax Antigens
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Nucleotide sequences encoding 203 AA (amino acids) of P. vivax merozoite surface protein 1 (rPvMSP1; Accession number AAA63427.1) and 259 AA of P.vivax circumsporozoite protein 1 (rPvCSP1; P08677.2) were cloned into expression vector pMAL-c2X (New England BioLabs, Ipswich, MA, USA). Both recombinant proteins were produced by Escherichia coli expression hosts and purified on amylose resin and DEAE Sepharose ® (GE healthcare, Uppsala, Sweden).
6
Recombinant Expression of Malaria Antigens
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Nucleotide constructs encoding 373 AA (amino acids) of P. falciparum MSP1 (Accession Number XP_001352170.1), 356 AA of P. ovale MSP1 (ACZ51239.1), and 350 AA of P. malariae MSP1 (ACZ51237.1) corresponding to the C-terminus region of the protein (42 kD) and a nucleotide construct encoding 448 AA of P. falciparum AMA1 (XP_001348015.1) were commercially synthesized with an Escherichia coli codon bias (Genscript, Piscataway, NJ, USA). Consensus sequences derived from the alignment of several strains for each species and each protein were used for the design of synthetic genes. All genes were inserted in an expression vector containing a Maltose binding protein fusion partner pMAL-c2X® (New England BioLabs, Ipswich, MA, USA), produced in E. coli expression hosts and purified on amylose resin and DEAEsepharose ®(GE healthcare, Uppsala, Sweden).
7
Purification of HeLa Cell Kinases
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HeLa cytoplasmic extracts (S100) and nuclear extracts (NE) were prepared following Dignam's protocol (42 (link)). These extracts were further separated on diethylaminoethyl (DEAE) Sepharose (GE Healthcare) for in vitro kinase assays. To identify the kinase responsible for phosphorylating the HP1α N-terminal domain, 10 mg of HeLa S100 or NE was loaded onto a HiTrap Q HP Column (GE Healthcare) equilibrated with buffer A (20 mM HEPES-KOH [pH 7.9], 10% glycerol, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride and 0.5 mM dithiothreitol [DTT]). The bound proteins were eluted step-wise with a gradient from 0 to 1 M KCl in buffer A. After the in vitro kinase assay, fractions with kinase activity were pooled and further separated on a Superose 6 Column (GE Healthcare) equilibrated with buffer A containing 150 mM KCl.
8
Purification and Synthesis of Bromoacrylic Compounds
9
Preparation and Characterization of Cysteine Sulfoxides
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Chemicals, solvents, pyridoxal-5′-phosphate (PLP), D,L-dithiothreitol (DTT), alliin, dialk(en)yldisulfides, amphotericin B (AmpB), fluconazole (FLC), 5-flucytosine (5-FC), kanamycin, YPD media and Sabouraud dextrose agar were purchased from Sigma-Aldrich (St. Louis, MO, USA); DEAE-sepharose was from “GE Healthcare” (Chicago, IL, USA); RPMI 1640 media and 3-(N-morpholino)propanesulfonic acid (MOPS) were purchased from PanEco (Moscow, Russia); S-alkyl-L-cysteine sulfoxides (methiin, ethiin and propiin) were synthesized according to the previously reported procedures [13 (link)]. 2-Nitro-5-thiobenzoate was obtained as described [30 ]. E. coli strain BL21 (DE3) F-ompT hsdSB gal dcm (DE3) was obtained from Novagen (Darmstadt, Germany). The plasmid with the gene of the C. freundii C115H MGL mutant form was obtained previously [13 (link)].
10
Isolation and Purification of Whole GAGs
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Whole GAGs were isolated from pancreata using methods described previously (43 (link)–45 (link)). Briefly, the samples were homogenized, defatted in acetone over two 24-hour periods at 4°C, and then subsequently digested in solution containing 2 mL 0.1 M Tris-HCl, pH 8.0, 2 mM CaCl2, 1% Triton X-100, and pronase (Roche) 0.8 mg/mL at 55°C. After 48 hours, the enzyme was inactivated by heating to 100°C for 15 min. The buffer was adjusted to 2 mM MgCl2, benzonase (Sigma; 100 mU) was added, and the sample was incubated for 2 h at 37°C. After inactivation of the enzyme (15 min, 100°C) any undigested material was removed by centrifugation for 1 h at 4000 g. The supernatant was applied to a DEAE-Sepharose (GE Healthcare; 2mL resin), washed with ~10 column volumes of loading buffer (~pH8 Tris Buffer, 0.1M NaCl). The sample was applied to the column, reapplied, and was washed with loading buffer. The sample was then eluted in 3CVs of elution buffer (~pH8 Tris Buffer, 2M NaCl), and desalted with a commercial PD-10 column (GE Healthcare, Chicago, IL).
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