Conjugated 17-AAG or/and CP-Sepharose 4B were prepared by incubating 17-AAG or/and CP with cyanogen bromide (CNBr)-activated Sepharose 4B (GE Healthcare, Sweden), and the pulldown assay was carried out as described [36 (link)].
Sepharose 4b
Manufactured by GE Healthcare
Sourced in United States, Sweden, United Kingdom, Germany
Sepharose 4B is a cross-linked agarose-based gel matrix used as a chromatography medium for the purification and separation of biomolecules. It provides a high degree of chemical and physical stability, as well as a porous structure that facilitates efficient adsorption and elution of target molecules.
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Lab products found in correlation
Ethylene diamine tetraacetic acid (edta), by Merck Group (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
7 methyl gtp sepharose 4b, by GE Healthcare (2 mentions)
Immobilon ipvh00010, by Merck Group (2 mentions)
Fast flow 16 266, by Merck Group (2 mentions)
Iodoacetamide solution, by Merck Group (2 mentions)
Methoxy peg30k amine, by Laysan Bio (2 mentions)
Hepes, by Merck Group (2 mentions)
Octyl β d glucopyranoside, by Dojindo Laboratories (2 mentions)
Glutathione sepharose 4b, by GE Healthcare (2 mentions)
Deae sepharose fast flow, by GE Healthcare (2 mentions)
Lysozyme, by Merck Group (2 mentions)
Fetal calf serum (fcs), by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (2 mentions)
G4251, by Merck Group (1 mentions)
96 protocols using sepharose 4b
1
Affinity Pulldown Assay for 17-AAG
2
Enrichment and Detection of Ubiquitinated Proteins
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For sample enrichment in ubiquitinated substrates cells were scraped off the dishes and collected in ice-cold PBS. Pellets were resuspended in 150 μl of lysis buffer (50 mM TRIS-HCl pH 7.4, 150 mM NaCl, 10 mM iodoacetamide, 2 mM PMSF, 20 mM Na3MoO4, 0.5% NP-40 and protease inhibitor cocktail). Lysates were briefly sonicated and centrifuged at 16,000 × g for 10 min. The samples were incubated overnight at 4 °C with 100 μg/ml of GST-TUBEs (LifeSensors, UM102). Thereafter, 30 μl of protein gluthatione–sepharose 4B (GE Healthcare, 17-0756-01) was added and incubations proceeded at 4 °C for 2 h. Beads were washed 3 times with lysis buffer and pulled-down proteins were eluted with 500 μl of lysis buffer supplemented with 10 mM of reduced gluthatione (Sigma-Aldrich, G4251). Samples were incubated with 2 μg of anti-HIF1A antibody overnight at 4 °C, 30 μl of protein G–Sepharose was added to the sample and incubations proceeded at 4 °C for 2 h. Beads were washed 3 times with lysis buffer, denatured with 2× Laemmli buffer and boiled at 100 °C. Samples were then analyzed by SDS-PAGE. The membranes were blocked with 5% non-fat milk in TBS-T and probed for the proteins of interest.
3
Purification of PQBP5 and Polyglutamine Proteins
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Full-length and deletion mutant PQBP5 and polyQ proteins were expressed in E. coli cells (#71400, Sigma Aldrich) by transformation of pET-28a-human PQBP5 and pET-28a-human PQBP5 deletion mutants or by transformation of pGEX-6P-1-AT1-33Q; pGEX-6P-1-AT1-86Q; pGEX-3X-Htt-17Q; and pGEX-3X-Htt-103Q98 (link), as described above. E. coli cells were collected by centrifugation, and His-tag PQBP5 fusion proteins were purified as described above. For GST-polyQ fusion proteins, the pellets were resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 1 mM DTT, 0.1% Triton X-100) containing 0.1 mg/ml lysozyme and protease inhibitor cocktail (#539134, Merck Millipore). After 30 min on ice, the cells were sonicated at level 6 for 10 min and centrifuged at 16,000 × g for 20 min at 4 °C. GST fusion proteins were added to glutathione Sepharose 4B (#17-0756-05, GE Healthcare, Chicago, IL, USA). After washing with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 1 mM DTT), the proteins were eluted with elution buffer (50 mM Tris-HCl (pH 8.0), 1% Triton X-100, 20 mM glutathione).
4
Affinity Purification of Hsp90 Proteins
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CNBr-activated Sepharose™4B (GE Healthcare) was swelled in 1mM HCl and washed with coupling buffer (0.1 M NaHCO3, 0.5M NaCl, pH = 8.3). After the resin was swelled, washed, and added to the coupling buffer, FM807 was dissolved in dimethyl sulfoxide (DMSO) and added to the resin (up to 10 μmoles per mL of medium). The mixture was rotated end over end for 4 h at room temperature, and excess ligand was removed by washing with coupling buffer. Any remaining active groups were blocked for 2 h at room temperature with the capping solution. The column was then equilibrated with coupling buffer for 1 h. Hsp90 test proteins were added to the resin, the mixture was rotated overnight at 4°C. Any unbound proteins were removed by washing buffer. Loading buffer was added to the resin, boiled for 10 min, and separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein separation was followed by western blotting for the protein of interest.
5
Affinity Capture of Protein Complexes
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Plant extracts (generated in co-immunoprecipitation buffer) were incubated with 50 µL of 7-methyl-GTP Sepharose® 4B GE Healthcare or Sepharose 4B (when stated) for 2 h at 4ºC. After washing, the retained proteins were eluted in 50 µL of 2x Laemmli buffer and subjected to SDS-PAGE. Experiments were done at least three times, obtaining similar results. Thus, a representative replicate is shown in the figures.
6
Passive Transfer of Lupus IgG1 in Mice
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First, mouse IgG1 was isolated from the sera of highly IgG1-ANA-positive aged lupus-prone MRL-lpr/lpr mice (n = 20; age ≥ 15 weeks) (lupus IgG1) or age-matched control C57BL/6J mice (n = 20) (control IgG1) by immunoaffinity column chromatography using goat anti-mouse IgG1 (Thermo Fisher Scientific, Inc., San Jose, CA, USA) coupled to CNBr-activated Sepharose™ 4B (GE Healthcare, Chicago, USA) and quantified using the Pierce™ BCA Protein Assay Kit. The homogeneity and reactivity of the IgG1 antibody mixtures were analyzed by SDS-PAGE and IF staining with HEp-2 cells.
Second, the experimental design and time lines were followed as shown inFigure 4A . Briefly, eighteen 10-week-old female MRL-lpr/lpr mice were divided equally into three groups which were defined as the non-treated group, control IgG1 group (treated with IgG1 from age-matched control mice), and lupus IgG1 group (treated with IgG1 from aged MRL-lpr/lpr mice). Purified IgG1 was administered intravenously via the tail vein at 1.0 mg/(mouse∙week) for 10–18 weeks of age. Samples (blood, urine, spleen, and kidney) were collected at the indicated time points. All mice were sacrificed at 19 weeks of age for sample collection.
Second, the experimental design and time lines were followed as shown in
7
Kinase Inhibitor Sepharose Conjugation
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Kinase inhibitors were conjugated with local laboratory generated ECH Sepharose 4B using the carbodiimide coupling method (12, 13 (link)). Briefly, nine kinase inhibitors (Palbociclib, Crizotinib, GSK690693, AZD4547, CZC-8004, Afatinib, FRAX597, Abemaciclib, and Axitinib; Supplementary Table S1 ) were separately conjugated to homemade ECH Sepharose 4B via carbodiimide coupling chemistry as described previously (12 (link)). ECH Sepharose 4B was synthesized using conjugating 6-Aminohexanoic acid (Sigma) to cyanogen bromide (CNBr)-activated Sepharose 4B (GE Healthcare). Each kinase inhibitor was dissolved in 50% dimethylformamide (DMF)/ethanol (EtOH) and added to the ECH Sepharose 4B beads in the presence of 0.1 mol/L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and allowed to react overnight at 4°C with rotation. After coupling, the unreacted groups were inactivated with ethanolamine. Subsequently, beads were washed with 0.1 mol/L Tris-HCl, pH 8.3 with 500 mmol/L NaCl and 0.1 mol/L acetate, pH 4.0 with 500 mmol/L NaCl and stored in 20% ethanol at 4°C in the dark.
8
Histone Extraction from Sea Cucumber
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High-purity reagents (SDS, Tris, different salts, bromophenol blue, EDTA, DTT, glycerol, urea, NH4HCO3, trifluoroacetic acid, and some other compounds) were obtained from Sigma (St. Louis, MO, USA). Histones H1, H2A, H2B, H3, and H4 of calf thymus were obtained from Sigma (product number 10223565001, St. Louis, MO, USA). Sepharose 4B was obtained from GE Healthcare Life Sciences (New York, NY, USA). Sea cucumber Eupentacta fraudatrix (holothurians) was collected from Peter the Great Bay in the Japan Sea. Samples of holothurians were frozen to −70 °C and stored until extract preparation.
9
Phage Purification and Negative Staining
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Prior to electron microscopy, phages were purified by gel filtration on Sepharose 4B (GE Healthcare). Phage suspension applied onto 200 mesh formvar-coated copper grids was negatively stained with 2% uranyl acetate and subsequently analyzed using a JEM-2100 (JEOL Ltd., Tokyo, Japan) transmission electron microscope.
10
Purification of CC11 Monoclonal Antibody
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Purified McAb CC11 (25 mg) and normal mouse IgG (11.2 mg) were coupled to CNBr-activated Sepharose 4B (GE, Beijing, China) as described by the manufacturer. Approximately 1010 bacteria culture (S. agalactiae strain TW3) were sonicated in 100 ml lysis buffer (20 mM Tris, 5 mM EDTA, 1% Triton X-100, pH 7.4) for 3 min at room temperature and incubated for 45 min at 4 °C. Cell debris was removed by centrifugation (30 min, 13,000×g, 4 °C). Prior to chromatography columns were equilibrated with 100 ml of wash buffer (50 mM Tris, 500 mM NaCl, 0.02% NaN3, 0.1%, Triton X-100, pH 8.0). The 100 ml lysate was passed through the mouse IgG column and then through the McAb CC11 column at a flow rate of 0.2 ml/min. Unbound proteins were removed by extensive washing with 150 ml wash buffer and 10 ml of 150 mM NaCl. McAb CC11-bound protein was eluted with 50 ml of acidic buffer (500 mM NaCl, 200 mM acetic acid, 0.1% Triton X-100, pH 2.8) at a flow rate of 0.5 ml/min. Columns were then equilibrated with 40 ml of 100 mM Tris-HCl buffer (pH 7.8) and 10 ml of 150 mM NaCl. Remaining protein was then eluted with 40 ml of alkaline buffer (50 mM diethylamine, 0.1% Triton X-100, pH 11.5). Fractions (4 ml) were collected and immediately neutralized (1 ml of 1 M Tris buffer, pH 9). Protein containing samples were subjected to SDS-PAGE. The eluted protein concentration was quantitated by standard BCA assay.
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