sepharose cl 4b  (Millipore)


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    Structured Review

    Millipore sepharose cl 4b
    Fractionation of PGs secreted from the MDA-MB-231 breast cancer cell line by anion-exchange and gel permeation chromatographies. One liter of conditioned medium was fractionated on a DEAE-Sephacel column (40 ml bed volume). The column was eluted stepwise with 3 volumes of the formamide buffer described under “Experimental Procedures” containing 0.2 M NaCl and 10 volumes of a NaCl linear gradient ranging from 0.2 to 1.0 M NaCl. Fractions of 6.7 ml were collected, and aliquots were precipitated by the addition of ethanol in the presence of potassium acetate. Precipitates were dissolved in distilled water, analyzed for their GAG content by the DMMB method (A), and subjected to SDS-PAGE analysis using a 4% stacking - 10% separating gel (B). Gels were stained with toluidine blue, followed by staining with coomassie blue. Serglycin was detected throughout the dissolved aliquots by Western blotting analysis after chondroitinase ABC digestion using a polyclonal antibody against serglycin (C) (SG, standard of serglycin used as positive control). A major PG peak was eluted from 0.50 to 0.7 M NaCl and pooled as indicated by the bar. Pooled serglycin was further fractionated by gel permeation chromatography (D). The pooled fractions were chromatographed on a <t>Sepharose</t> <t>CL-4B</t> column. The column was eluted with 4 M guanidine hydrochloride, 50 mM sodium acetate buffer, pH 5.8. Fractions were collected, and PGs were monitored by the DMMB method (D), were subjected to SDS-PAGE following toluidine blue and coomassie staining (E), and were analyzed by Western blotting after chondroitinase ABC digestion (F) (SG, standard of serglycin used as positive control).
    Sepharose Cl 4b, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/sepharose cl 4b/product/Millipore
    Average 99 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    sepharose cl 4b - by Bioz Stars, 2020-01
    99/100 stars

    Images

    1) Product Images from "Serglycin Is Implicated in the Promotion of Aggressive Phenotype of Breast Cancer Cells"

    Article Title: Serglycin Is Implicated in the Promotion of Aggressive Phenotype of Breast Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0078157

    Fractionation of PGs secreted from the MDA-MB-231 breast cancer cell line by anion-exchange and gel permeation chromatographies. One liter of conditioned medium was fractionated on a DEAE-Sephacel column (40 ml bed volume). The column was eluted stepwise with 3 volumes of the formamide buffer described under “Experimental Procedures” containing 0.2 M NaCl and 10 volumes of a NaCl linear gradient ranging from 0.2 to 1.0 M NaCl. Fractions of 6.7 ml were collected, and aliquots were precipitated by the addition of ethanol in the presence of potassium acetate. Precipitates were dissolved in distilled water, analyzed for their GAG content by the DMMB method (A), and subjected to SDS-PAGE analysis using a 4% stacking - 10% separating gel (B). Gels were stained with toluidine blue, followed by staining with coomassie blue. Serglycin was detected throughout the dissolved aliquots by Western blotting analysis after chondroitinase ABC digestion using a polyclonal antibody against serglycin (C) (SG, standard of serglycin used as positive control). A major PG peak was eluted from 0.50 to 0.7 M NaCl and pooled as indicated by the bar. Pooled serglycin was further fractionated by gel permeation chromatography (D). The pooled fractions were chromatographed on a Sepharose CL-4B column. The column was eluted with 4 M guanidine hydrochloride, 50 mM sodium acetate buffer, pH 5.8. Fractions were collected, and PGs were monitored by the DMMB method (D), were subjected to SDS-PAGE following toluidine blue and coomassie staining (E), and were analyzed by Western blotting after chondroitinase ABC digestion (F) (SG, standard of serglycin used as positive control).
    Figure Legend Snippet: Fractionation of PGs secreted from the MDA-MB-231 breast cancer cell line by anion-exchange and gel permeation chromatographies. One liter of conditioned medium was fractionated on a DEAE-Sephacel column (40 ml bed volume). The column was eluted stepwise with 3 volumes of the formamide buffer described under “Experimental Procedures” containing 0.2 M NaCl and 10 volumes of a NaCl linear gradient ranging from 0.2 to 1.0 M NaCl. Fractions of 6.7 ml were collected, and aliquots were precipitated by the addition of ethanol in the presence of potassium acetate. Precipitates were dissolved in distilled water, analyzed for their GAG content by the DMMB method (A), and subjected to SDS-PAGE analysis using a 4% stacking - 10% separating gel (B). Gels were stained with toluidine blue, followed by staining with coomassie blue. Serglycin was detected throughout the dissolved aliquots by Western blotting analysis after chondroitinase ABC digestion using a polyclonal antibody against serglycin (C) (SG, standard of serglycin used as positive control). A major PG peak was eluted from 0.50 to 0.7 M NaCl and pooled as indicated by the bar. Pooled serglycin was further fractionated by gel permeation chromatography (D). The pooled fractions were chromatographed on a Sepharose CL-4B column. The column was eluted with 4 M guanidine hydrochloride, 50 mM sodium acetate buffer, pH 5.8. Fractions were collected, and PGs were monitored by the DMMB method (D), were subjected to SDS-PAGE following toluidine blue and coomassie staining (E), and were analyzed by Western blotting after chondroitinase ABC digestion (F) (SG, standard of serglycin used as positive control).

    Techniques Used: Fractionation, Multiple Displacement Amplification, SDS Page, Staining, Western Blot, Positive Control, GPC Assay

    Related Articles

    Centrifugation:

    Article Title: Klf5 regulates muscle differentiation by directly targeting muscle-specific genes in cooperation with MyoD in mice
    Article Snippet: The chromatin was then pre-cleared by incubating with Sepharose CL-4B (Sigma- Aldrich) for 2 hr at 4°C with constant rotation. .. After centrifugation, anti-MyoD or anti-Klf5 antibody was added and rotated overnight at 4°C.

    Article Title: Lectin microarray and mass spectrometric analysis of hepatitis C proteins reveals N-linked glycosylation
    Article Snippet: The resulting polypeptides were collected by centrifugation and further digested with PNGase F (New England BioLabs, Ipswich, MA) overnight at 37°C. .. The released N-linked glycans were collected and desalted using Sepharose CL-4B (Sigma–Aldrich) and MS analysis of the N-linked glycans of HCVcc was conducted by MALDI-TOF/TOF-MS (UltrafleXtreme, Bruker Daltonics, Bremen, Germany) according to a previously described protocol.

    Mass Spectrometry:

    Article Title: Lectin microarray and mass spectrometric analysis of hepatitis C proteins reveals N-linked glycosylation
    Article Snippet: .. The released N-linked glycans were collected and desalted using Sepharose CL-4B (Sigma–Aldrich) and MS analysis of the N-linked glycans of HCVcc was conducted by MALDI-TOF/TOF-MS (UltrafleXtreme, Bruker Daltonics, Bremen, Germany) according to a previously described protocol. .. [ ] Measurements were obtained in positive ion mode, and mass-to-charge ratio (m/z) data were annotated using GlycoWorkbench software.

    Stable Transfection:

    Article Title: How ligand binds to the type 1 insulin-like growth factor receptor
    Article Snippet: A CHO Lec8 cell line stably expressing IGF-1RΔβ (a construct of the human IGF-1R ectodomain comprising residues 1–905 but with the highly glycosylated segment (residues 718–741) near the N terminus of the β chain replaced by the quadruplet AGNN) was originally obtained from CSIRO (Parkville, Australia) by the corresponding author’s laboratory . .. Typically, 10 L conditioned medium containing IGF-1RΔβ was pumped through a 50 mL column of Sepharose CL-4B (Sigma-Aldrich) to remove non-specifically binding material and then a 50 mL affinity column at 4 °C, followed by extensive column washing to remove unbound protein.

    Construct:

    Article Title: How ligand binds to the type 1 insulin-like growth factor receptor
    Article Snippet: A CHO Lec8 cell line stably expressing IGF-1RΔβ (a construct of the human IGF-1R ectodomain comprising residues 1–905 but with the highly glycosylated segment (residues 718–741) near the N terminus of the β chain replaced by the quadruplet AGNN) was originally obtained from CSIRO (Parkville, Australia) by the corresponding author’s laboratory . .. Typically, 10 L conditioned medium containing IGF-1RΔβ was pumped through a 50 mL column of Sepharose CL-4B (Sigma-Aldrich) to remove non-specifically binding material and then a 50 mL affinity column at 4 °C, followed by extensive column washing to remove unbound protein.

    Incubation:

    Article Title: Klf5 regulates muscle differentiation by directly targeting muscle-specific genes in cooperation with MyoD in mice
    Article Snippet: The pellet was further lysed in ice-cold RIPA buffer and incubated on ice for 10 min. .. The chromatin was then pre-cleared by incubating with Sepharose CL-4B (Sigma- Aldrich) for 2 hr at 4°C with constant rotation.

    Article Title: Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting
    Article Snippet: EV/dye mixtures were incubated for 1 h at 37°C and stored at 4°C until purification (maximally 16 h). .. For removal of unincorporated label, Sepharose CL-4B (Sigma-Aldrich) was packed in a XK-16/20 column (GE Healthcare) according to the manufacturer's instructions.

    Article Title: Norbin Stimulates the Catalytic Activity and Plasma Membrane Localization of the Guanine-Nucleotide Exchange Factor P-Rex1 *
    Article Snippet: The next day, fractions were pre-cleared for 1 h with 100 μl of Sepharose CL-4B (Sigma) to minimize nonspecific protein binding to beads later on. .. Each cytosol fraction was then split in two, and one-half incubated with 10 μg of purified recombinant human full-length EE-P Rex1 protein that had been immobilized using EE antibody covalently coupled to protein G-Sepharose , the other half with EE-antibody beads alone (without EE-P-Rex1), as a control.

    Article Title: SLAB51 Probiotic Formulation Activates SIRT1 Pathway Promoting Antioxidant and Neuroprotective Effects in an AD Mouse Model
    Article Snippet: .. In detail, aliquots of brain homogenates were incubated with agarose-conjugated protein A immobilized on Sepharose CL-4B (Sigma-Aldrich S.r.L., Milano, Italy), followed by SDS-PAGE and immunoblotting. ..

    Article Title: Proto-oncogene Activity of Melanoma Antigen-A11 (MAGE-A11) Regulates Retinoblastoma-related p107 and E2F1 Proteins *
    Article Snippet: Samples were precleared for 15 min at 4 °C using Sepharose CL-4B (Sigma) and immunoprecipitated for 2 h at 4 °C using anti-FLAG M2-agarose affinity resin ( ). .. For phosphorylation experiments, 30 μg of cell extract protein in 40 μl of immunoprecipitation buffer without NaF, sodium vanadate, EDTA, or deoxycholate was incubated for 1 h at 4 °C with or without 4000 IU λ protein phosphatase (New England Biolabs), according to the instructions of the manufacturer ( ).

    Article Title: Sec24C/D-isoform–specific sorting of the preassembled ER–Golgi Q-SNARE complex
    Article Snippet: SNARE-GST binding assay For the GST binding assay, 50% (vol/vol) slurry of glutathione Sepharose 4 fast was mixed 1:1 with 50% (vol/vol) slurry of Sepharose CL-4B (Sigma-Aldrich). .. GST or SNARE-GST fusion proteins (100–300 μg, to obtain equal amounts of fusion protein binding) were incubated with 20 μl of the 50% (vol/vol) mixed slurry in assay buffer (25 mM HEPES, pH 7.2 [KOH], 150 mM KOAc, 2 mM MgOAc, 0.02% [vol/vol] monothioglycerol) in a total volume of 1 ml for 1 h at 4°C.

    GPC Assay:

    Article Title: Serglycin Is Implicated in the Promotion of Aggressive Phenotype of Breast Cancer Cells
    Article Snippet: .. The major PG population eluting with 0.55–0.7 M NaCl was pooled, concentrated with Vivaspin 20 Ultrafiltration devices (Sartorius Biotech) and subjected to gel permeation chromatography on a Sepharose CL-4B (Sigma-Aldrich) column. ..

    Expressing:

    Article Title: Proto-oncogene Activity of Melanoma Antigen-A11 (MAGE-A11) Regulates Retinoblastoma-related p107 and E2F1 Proteins *
    Article Snippet: Paragraph title: Expression Studies ... Samples were precleared for 15 min at 4 °C using Sepharose CL-4B (Sigma) and immunoprecipitated for 2 h at 4 °C using anti-FLAG M2-agarose affinity resin ( ).

    Article Title: How ligand binds to the type 1 insulin-like growth factor receptor
    Article Snippet: Paragraph title: Expression and purification of IGF-1RΔβ ... Typically, 10 L conditioned medium containing IGF-1RΔβ was pumped through a 50 mL column of Sepharose CL-4B (Sigma-Aldrich) to remove non-specifically binding material and then a 50 mL affinity column at 4 °C, followed by extensive column washing to remove unbound protein.

    Modification:

    Article Title: How ligand binds to the type 1 insulin-like growth factor receptor
    Article Snippet: Cells were thawed into Dulbecco's modified Eagle's medium F12 + GlutaMAX medium (Life Technologies) containing 10 μg mL−1 puromycin (Life Technologies) plus 10% fetal bovine serum (Life Technologies) and expanded by passaging several times in T150 tissue culture flasks at 37 °C/5% CO2 . .. Typically, 10 L conditioned medium containing IGF-1RΔβ was pumped through a 50 mL column of Sepharose CL-4B (Sigma-Aldrich) to remove non-specifically binding material and then a 50 mL affinity column at 4 °C, followed by extensive column washing to remove unbound protein.

    Western Blot:

    Article Title: Proto-oncogene Activity of Melanoma Antigen-A11 (MAGE-A11) Regulates Retinoblastoma-related p107 and E2F1 Proteins *
    Article Snippet: Immunoblots of extracts from COS1 cells (2 × 106 cells/10-cm dish, 7 × 105 cells/6-cm dish) transfected using DEAE-dextran were prepared in immunoblot lysis buffer containing 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 0.15 m NaCl, 2 m m EDTA, 50 m m NaF, 2 m m sodium vanadate, 50 m m Tris-HCl (pH 7.5), 1 m m phenylmethylsulfonyl fluoride, 1 m m dithiothreitol, and protease inhibitors (Roche Applied Science). .. Samples were precleared for 15 min at 4 °C using Sepharose CL-4B (Sigma) and immunoprecipitated for 2 h at 4 °C using anti-FLAG M2-agarose affinity resin ( ).

    Article Title: Serglycin Is Implicated in the Promotion of Aggressive Phenotype of Breast Cancer Cells
    Article Snippet: Serglycin was detected throughout the dissolved aliquots with Western blotting analysis as described above. .. The major PG population eluting with 0.55–0.7 M NaCl was pooled, concentrated with Vivaspin 20 Ultrafiltration devices (Sartorius Biotech) and subjected to gel permeation chromatography on a Sepharose CL-4B (Sigma-Aldrich) column.

    Article Title: Inhibition of plasminogen activation by apo(a): role of carboxyl-terminal lysines and identification of inhibitory domains in apo(a) [S]
    Article Snippet: The sample was then subjected to gel-filtration chromatography over Sepharose CL-4B (Sigma-Aldrich) (2.5 cm × 80 cm column) using buffer A containing 0.1% (v/v) Tween 20 and 0.1 M proline. .. Fractions were collected, and samples with an absorbance over 0.1 at 280 nm were subjected to Western blot analysis using an anti-apo(a) antibody to determine Lp(a)-containing fractions.

    Flow Cytometry:

    Article Title: Klf5 regulates muscle differentiation by directly targeting muscle-specific genes in cooperation with MyoD in mice
    Article Snippet: The chromatin was then pre-cleared by incubating with Sepharose CL-4B (Sigma- Aldrich) for 2 hr at 4°C with constant rotation. .. Protein A-Sepharose 4B fast flow (BD) was then added and incubated for 1 hr at 4°C with constant rotation.

    Article Title: Norbin Stimulates the Catalytic Activity and Plasma Membrane Localization of the Guanine-Nucleotide Exchange Factor P-Rex1 *
    Article Snippet: Fifteen fractions were collected, ranging from the flow-through to the 1 m NaCl fraction and from 10 to 50 ml in volume, depending on the protein profile. .. The next day, fractions were pre-cleared for 1 h with 100 μl of Sepharose CL-4B (Sigma) to minimize nonspecific protein binding to beads later on.

    Article Title: Inhibition of plasminogen activation by apo(a): role of carboxyl-terminal lysines and identification of inhibitory domains in apo(a) [S]
    Article Snippet: The sample was then subjected to gel-filtration chromatography over Sepharose CL-4B (Sigma-Aldrich) (2.5 cm × 80 cm column) using buffer A containing 0.1% (v/v) Tween 20 and 0.1 M proline. .. Samples containing Lp(a) were pooled and diluted 3-fold with distilled water and loaded onto a DEAE-Sepharose Fast Flow (Pharmacia) ion exchange column (2.5 × 3 cm column).

    Chromatography:

    Article Title: Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting
    Article Snippet: For removal of unincorporated label, Sepharose CL-4B (Sigma-Aldrich) was packed in a XK-16/20 column (GE Healthcare) according to the manufacturer's instructions. .. Column was connected to a refrigerated ÄKTA pure chromatography system, equilibrated with PBS, and EV/dye mixtures were injected.

    Article Title: Inhibition of plasminogen activation by apo(a): role of carboxyl-terminal lysines and identification of inhibitory domains in apo(a) [S]
    Article Snippet: .. The sample was then subjected to gel-filtration chromatography over Sepharose CL-4B (Sigma-Aldrich) (2.5 cm × 80 cm column) using buffer A containing 0.1% (v/v) Tween 20 and 0.1 M proline. .. Fractions were collected, and samples with an absorbance over 0.1 at 280 nm were subjected to Western blot analysis using an anti-apo(a) antibody to determine Lp(a)-containing fractions.

    Concentration Assay:

    Article Title: Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting
    Article Snippet: CellTracker Deep Red dye was dissolved at 2 mM concentration in dimethyl sulfoxide (DMSO) and mixed with fixed EV amounts (based on protein concentration) at a final dye concentration of 16 µM. .. For removal of unincorporated label, Sepharose CL-4B (Sigma-Aldrich) was packed in a XK-16/20 column (GE Healthcare) according to the manufacturer's instructions.

    Article Title: Norbin Stimulates the Catalytic Activity and Plasma Membrane Localization of the Guanine-Nucleotide Exchange Factor P-Rex1 *
    Article Snippet: The salt concentration of each fraction was adjusted to 150 mm by the addition of 3 m NaCl or by dilution with column buffer 3, as appropriate, and stored at 4 °C overnight. .. The next day, fractions were pre-cleared for 1 h with 100 μl of Sepharose CL-4B (Sigma) to minimize nonspecific protein binding to beads later on.

    Article Title: Monitoring Phospholipase A2 Activity with Gd-encapsulated Phospholipid Liposomes
    Article Snippet: Briefly, a lipid stock solution containing 90% DPPC/10% DSPE-PEG2000 (molar ratio) was dissolved in chloroform and subsequently dried using a direct stream of nitrogen prior to vacuum desiccation for a minimum of 4 h. The resultant dried lipid films were rehydrated with 0.5 M concentration of Gadoteridol or 100 mM carboxyfluorescein (CF) for 30 min. .. Nonentrapped compound was removed via centrifugal filter devices (Amicon Ultra-4, 50 000 MWCO) and size exclusion chromatography using Sepharose CL-4B (Sigma-Aldrich).

    Article Title: Inhibition of plasminogen activation by apo(a): role of carboxyl-terminal lysines and identification of inhibitory domains in apo(a) [S]
    Article Snippet: PMSF was added to a final concentration of 0.1 mM, and the plasma was rotated at 4°C for 15 min. .. The sample was then subjected to gel-filtration chromatography over Sepharose CL-4B (Sigma-Aldrich) (2.5 cm × 80 cm column) using buffer A containing 0.1% (v/v) Tween 20 and 0.1 M proline.

    Northern Blot:

    Article Title: Vitamin C-driven epirubicin loading into liposomes
    Article Snippet: Materials Hydrogenated soy phosphatidylcholine (HSPC), 1,2-dis tearoyl-sn -glycero-phosphoethanolamine-N -(poly[ethylen e glycol]2000) (DSPE-PEG 2000) and cholesterol (Chol) were purchased from Northern Lipids (Vancouver, BC, Canada). .. Sephadex G-50 fine and Sepharose CL-4B were obtained from Sigma-Aldrich (Poznan, Poland).

    Generated:

    Article Title: Lectin microarray and mass spectrometric analysis of hepatitis C proteins reveals N-linked glycosylation
    Article Snippet: The released N-linked glycans were collected and desalted using Sepharose CL-4B (Sigma–Aldrich) and MS analysis of the N-linked glycans of HCVcc was conducted by MALDI-TOF/TOF-MS (UltrafleXtreme, Bruker Daltonics, Bremen, Germany) according to a previously described protocol. .. The relative intensity was analyzed and generated using FlexAnalysis software (Bruker Daltonics) based on the MALDI-TOF-MS intensity.

    Protein Concentration:

    Article Title: Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting
    Article Snippet: CellTracker Deep Red dye was dissolved at 2 mM concentration in dimethyl sulfoxide (DMSO) and mixed with fixed EV amounts (based on protein concentration) at a final dye concentration of 16 µM. .. For removal of unincorporated label, Sepharose CL-4B (Sigma-Aldrich) was packed in a XK-16/20 column (GE Healthcare) according to the manufacturer's instructions.

    Sequencing:

    Article Title: Lectin microarray and mass spectrometric analysis of hepatitis C proteins reveals N-linked glycosylation
    Article Snippet: The ultrafiltration retentate was digested with sequencing-grade trypsin (Promega, Madison, WI) overnight at 37°C. .. The released N-linked glycans were collected and desalted using Sepharose CL-4B (Sigma–Aldrich) and MS analysis of the N-linked glycans of HCVcc was conducted by MALDI-TOF/TOF-MS (UltrafleXtreme, Bruker Daltonics, Bremen, Germany) according to a previously described protocol.

    Sonication:

    Article Title: Klf5 regulates muscle differentiation by directly targeting muscle-specific genes in cooperation with MyoD in mice
    Article Snippet: The resultant cell lysate was sonicated using an ultrasonicator (output 50, 30-s pulses then 1-min pause × 13 times; UD-100: TOMY Seiko, Tokyo, Japan). .. The chromatin was then pre-cleared by incubating with Sepharose CL-4B (Sigma- Aldrich) for 2 hr at 4°C with constant rotation.

    Injection:

    Article Title: Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting
    Article Snippet: For removal of unincorporated label, Sepharose CL-4B (Sigma-Aldrich) was packed in a XK-16/20 column (GE Healthcare) according to the manufacturer's instructions. .. Column was connected to a refrigerated ÄKTA pure chromatography system, equilibrated with PBS, and EV/dye mixtures were injected.

    Binding Assay:

    Article Title: How ligand binds to the type 1 insulin-like growth factor receptor
    Article Snippet: .. Typically, 10 L conditioned medium containing IGF-1RΔβ was pumped through a 50 mL column of Sepharose CL-4B (Sigma-Aldrich) to remove non-specifically binding material and then a 50 mL affinity column at 4 °C, followed by extensive column washing to remove unbound protein. ..

    Article Title: Sec24C/D-isoform–specific sorting of the preassembled ER–Golgi Q-SNARE complex
    Article Snippet: .. SNARE-GST binding assay For the GST binding assay, 50% (vol/vol) slurry of glutathione Sepharose 4 fast was mixed 1:1 with 50% (vol/vol) slurry of Sepharose CL-4B (Sigma-Aldrich). .. GST or SNARE-GST fusion proteins (100–300 μg, to obtain equal amounts of fusion protein binding) were incubated with 20 μl of the 50% (vol/vol) mixed slurry in assay buffer (25 mM HEPES, pH 7.2 [KOH], 150 mM KOAc, 2 mM MgOAc, 0.02% [vol/vol] monothioglycerol) in a total volume of 1 ml for 1 h at 4°C.

    Fluorescence:

    Article Title: Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting
    Article Snippet: For removal of unincorporated label, Sepharose CL-4B (Sigma-Aldrich) was packed in a XK-16/20 column (GE Healthcare) according to the manufacturer's instructions. .. To determine whether labelling efficiency differed among EV samples, protein concentrations were measured using a MicroBCA Protein Assay, and fluorescence of 60 µL samples in a black 96-well plate was determined using a SpectraMax M2e microplate reader (Molecular Devices) at 630 nm excitation and 650 nm emission.

    Article Title: Preparation of Benzothiazolyl-Decorated Nanoliposomes
    Article Snippet: Cholesterol (Chol), SephadexG50 (course), calcein, Sepharose CL-4B were purchased from Sigma-Aldrich OM, Athens, Greece. .. Fluorescence intensity (FI) of samples was measured with a Shimatzu RF-5301PC spectrofluorophotometer (Kyoto, Japan) at 37 ± 0.1 °C.

    Passaging:

    Article Title: How ligand binds to the type 1 insulin-like growth factor receptor
    Article Snippet: Cells were thawed into Dulbecco's modified Eagle's medium F12 + GlutaMAX medium (Life Technologies) containing 10 μg mL−1 puromycin (Life Technologies) plus 10% fetal bovine serum (Life Technologies) and expanded by passaging several times in T150 tissue culture flasks at 37 °C/5% CO2 . .. Typically, 10 L conditioned medium containing IGF-1RΔβ was pumped through a 50 mL column of Sepharose CL-4B (Sigma-Aldrich) to remove non-specifically binding material and then a 50 mL affinity column at 4 °C, followed by extensive column washing to remove unbound protein.

    Isolation:

    Article Title: Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting
    Article Snippet: EV labelling and purification For functional assays, EVs were labelled with CellTracker Deep Red immediately after isolation. .. For removal of unincorporated label, Sepharose CL-4B (Sigma-Aldrich) was packed in a XK-16/20 column (GE Healthcare) according to the manufacturer's instructions.

    Article Title: Norbin Stimulates the Catalytic Activity and Plasma Membrane Localization of the Guanine-Nucleotide Exchange Factor P-Rex1 *
    Article Snippet: Paragraph title: Isolation of Norbin from Mouse Brain Cytosol Fractions with Recombinant EE-P-Rex1 ... The next day, fractions were pre-cleared for 1 h with 100 μl of Sepharose CL-4B (Sigma) to minimize nonspecific protein binding to beads later on.

    Article Title: Serglycin Is Implicated in the Promotion of Aggressive Phenotype of Breast Cancer Cells
    Article Snippet: Paragraph title: Isolation of PGs by DEAE-Sephacel and Sepharose CL-4B ... The major PG population eluting with 0.55–0.7 M NaCl was pooled, concentrated with Vivaspin 20 Ultrafiltration devices (Sartorius Biotech) and subjected to gel permeation chromatography on a Sepharose CL-4B (Sigma-Aldrich) column.

    Article Title: Inhibition of plasminogen activation by apo(a): role of carboxyl-terminal lysines and identification of inhibitory domains in apo(a) [S]
    Article Snippet: Paragraph title: Isolation of Lp(a) ... The sample was then subjected to gel-filtration chromatography over Sepharose CL-4B (Sigma-Aldrich) (2.5 cm × 80 cm column) using buffer A containing 0.1% (v/v) Tween 20 and 0.1 M proline.

    shRNA:

    Article Title: Proto-oncogene Activity of Melanoma Antigen-A11 (MAGE-A11) Regulates Retinoblastoma-related p107 and E2F1 Proteins *
    Article Snippet: Samples were precleared for 15 min at 4 °C using Sepharose CL-4B (Sigma) and immunoprecipitated for 2 h at 4 °C using anti-FLAG M2-agarose affinity resin ( ). .. Endogenous expression of AR and MAGE-A11 was inhibited in LAPC-4 cells using lentivirus shRNA prepared using the Open Biosystems TRC1 shRNA library.

    Transfection:

    Article Title: Proto-oncogene Activity of Melanoma Antigen-A11 (MAGE-A11) Regulates Retinoblastoma-related p107 and E2F1 Proteins *
    Article Snippet: Immunoblots of extracts from COS1 cells (2 × 106 cells/10-cm dish, 7 × 105 cells/6-cm dish) transfected using DEAE-dextran were prepared in immunoblot lysis buffer containing 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 0.15 m NaCl, 2 m m EDTA, 50 m m NaF, 2 m m sodium vanadate, 50 m m Tris-HCl (pH 7.5), 1 m m phenylmethylsulfonyl fluoride, 1 m m dithiothreitol, and protease inhibitors (Roche Applied Science). .. Samples were precleared for 15 min at 4 °C using Sepharose CL-4B (Sigma) and immunoprecipitated for 2 h at 4 °C using anti-FLAG M2-agarose affinity resin ( ).

    Purification:

    Article Title: Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting
    Article Snippet: Paragraph title: EV labelling and purification ... For removal of unincorporated label, Sepharose CL-4B (Sigma-Aldrich) was packed in a XK-16/20 column (GE Healthcare) according to the manufacturer's instructions.

    Article Title: Norbin Stimulates the Catalytic Activity and Plasma Membrane Localization of the Guanine-Nucleotide Exchange Factor P-Rex1 *
    Article Snippet: The next day, fractions were pre-cleared for 1 h with 100 μl of Sepharose CL-4B (Sigma) to minimize nonspecific protein binding to beads later on. .. Each cytosol fraction was then split in two, and one-half incubated with 10 μg of purified recombinant human full-length EE-P Rex1 protein that had been immobilized using EE antibody covalently coupled to protein G-Sepharose , the other half with EE-antibody beads alone (without EE-P-Rex1), as a control.

    Article Title: How ligand binds to the type 1 insulin-like growth factor receptor
    Article Snippet: Paragraph title: Expression and purification of IGF-1RΔβ ... Typically, 10 L conditioned medium containing IGF-1RΔβ was pumped through a 50 mL column of Sepharose CL-4B (Sigma-Aldrich) to remove non-specifically binding material and then a 50 mL affinity column at 4 °C, followed by extensive column washing to remove unbound protein.

    Article Title: Lectin microarray and mass spectrometric analysis of hepatitis C proteins reveals N-linked glycosylation
    Article Snippet: The envelope protein of purified HCVcc was digested with glycosidase as described in the Supplementary Materials and Methods sections. .. The released N-linked glycans were collected and desalted using Sepharose CL-4B (Sigma–Aldrich) and MS analysis of the N-linked glycans of HCVcc was conducted by MALDI-TOF/TOF-MS (UltrafleXtreme, Bruker Daltonics, Bremen, Germany) according to a previously described protocol.

    Affinity Chromatography:

    Article Title: How ligand binds to the type 1 insulin-like growth factor receptor
    Article Snippet: IGF-1RΔβ was recovered from harvested media by IGF-I affinity chromatography on a column of immobilized LONG-R3-IGF-I (GroPep Bioreagents; Australia) and then further purified by size-exclusion chromatography (SEC) as follows: the LONG-R3-IGF-1 affinity column was prepared by covalently binding 40 mg of media grade LONG-R3-IGF-I (GroPep Bioreagents; Australia) to 50 mL Mini-Leak Medium Agarose (Kem-En-Tec; Denmark) as per the manufacturer’s instructions. .. Typically, 10 L conditioned medium containing IGF-1RΔβ was pumped through a 50 mL column of Sepharose CL-4B (Sigma-Aldrich) to remove non-specifically binding material and then a 50 mL affinity column at 4 °C, followed by extensive column washing to remove unbound protein.

    Affinity Column:

    Article Title: How ligand binds to the type 1 insulin-like growth factor receptor
    Article Snippet: .. Typically, 10 L conditioned medium containing IGF-1RΔβ was pumped through a 50 mL column of Sepharose CL-4B (Sigma-Aldrich) to remove non-specifically binding material and then a 50 mL affinity column at 4 °C, followed by extensive column washing to remove unbound protein. ..

    CRISPR:

    Article Title: Klf5 regulates muscle differentiation by directly targeting muscle-specific genes in cooperation with MyoD in mice
    Article Snippet: For MyoD ChIP in CRISPR-engineered cells, cells differentiated for 3 days were used. .. The chromatin was then pre-cleared by incubating with Sepharose CL-4B (Sigma- Aldrich) for 2 hr at 4°C with constant rotation.

    Silver Staining:

    Article Title: Norbin Stimulates the Catalytic Activity and Plasma Membrane Localization of the Guanine-Nucleotide Exchange Factor P-Rex1 *
    Article Snippet: The next day, fractions were pre-cleared for 1 h with 100 μl of Sepharose CL-4B (Sigma) to minimize nonspecific protein binding to beads later on. .. Protein was denatured in boiling Laemmli sample buffer, resolved on 8% SDS-PAGE gels, and stained using the SilverQuestTM silver staining kit (Invitrogen).

    Chromatin Immunoprecipitation:

    Article Title: Klf5 regulates muscle differentiation by directly targeting muscle-specific genes in cooperation with MyoD in mice
    Article Snippet: Paragraph title: Chromatin immunoprecipitation (ChIP) ... The chromatin was then pre-cleared by incubating with Sepharose CL-4B (Sigma- Aldrich) for 2 hr at 4°C with constant rotation.

    SDS Page:

    Article Title: Norbin Stimulates the Catalytic Activity and Plasma Membrane Localization of the Guanine-Nucleotide Exchange Factor P-Rex1 *
    Article Snippet: The next day, fractions were pre-cleared for 1 h with 100 μl of Sepharose CL-4B (Sigma) to minimize nonspecific protein binding to beads later on. .. Protein was denatured in boiling Laemmli sample buffer, resolved on 8% SDS-PAGE gels, and stained using the SilverQuestTM silver staining kit (Invitrogen).

    Article Title: SLAB51 Probiotic Formulation Activates SIRT1 Pathway Promoting Antioxidant and Neuroprotective Effects in an AD Mouse Model
    Article Snippet: .. In detail, aliquots of brain homogenates were incubated with agarose-conjugated protein A immobilized on Sepharose CL-4B (Sigma-Aldrich S.r.L., Milano, Italy), followed by SDS-PAGE and immunoblotting. ..

    Article Title: Sec24C/D-isoform–specific sorting of the preassembled ER–Golgi Q-SNARE complex
    Article Snippet: SNARE-GST binding assay For the GST binding assay, 50% (vol/vol) slurry of glutathione Sepharose 4 fast was mixed 1:1 with 50% (vol/vol) slurry of Sepharose CL-4B (Sigma-Aldrich). .. Subsequently, glutathione Sepharose beads were washed three times with 750 μl of assay buffer to remove unbound material, and bound His6-Sec23A/24C was eluted with SDS–PAGE sample buffer.

    Software:

    Article Title: Lectin microarray and mass spectrometric analysis of hepatitis C proteins reveals N-linked glycosylation
    Article Snippet: The released N-linked glycans were collected and desalted using Sepharose CL-4B (Sigma–Aldrich) and MS analysis of the N-linked glycans of HCVcc was conducted by MALDI-TOF/TOF-MS (UltrafleXtreme, Bruker Daltonics, Bremen, Germany) according to a previously described protocol. .. [ ] Measurements were obtained in positive ion mode, and mass-to-charge ratio (m/z) data were annotated using GlycoWorkbench software.

    Functional Assay:

    Article Title: Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting
    Article Snippet: EV labelling and purification For functional assays, EVs were labelled with CellTracker Deep Red immediately after isolation. .. For removal of unincorporated label, Sepharose CL-4B (Sigma-Aldrich) was packed in a XK-16/20 column (GE Healthcare) according to the manufacturer's instructions.

    Recombinant:

    Article Title: Norbin Stimulates the Catalytic Activity and Plasma Membrane Localization of the Guanine-Nucleotide Exchange Factor P-Rex1 *
    Article Snippet: Paragraph title: Isolation of Norbin from Mouse Brain Cytosol Fractions with Recombinant EE-P-Rex1 ... The next day, fractions were pre-cleared for 1 h with 100 μl of Sepharose CL-4B (Sigma) to minimize nonspecific protein binding to beads later on.

    Size-exclusion Chromatography:

    Article Title: How ligand binds to the type 1 insulin-like growth factor receptor
    Article Snippet: IGF-1RΔβ was recovered from harvested media by IGF-I affinity chromatography on a column of immobilized LONG-R3-IGF-I (GroPep Bioreagents; Australia) and then further purified by size-exclusion chromatography (SEC) as follows: the LONG-R3-IGF-1 affinity column was prepared by covalently binding 40 mg of media grade LONG-R3-IGF-I (GroPep Bioreagents; Australia) to 50 mL Mini-Leak Medium Agarose (Kem-En-Tec; Denmark) as per the manufacturer’s instructions. .. Typically, 10 L conditioned medium containing IGF-1RΔβ was pumped through a 50 mL column of Sepharose CL-4B (Sigma-Aldrich) to remove non-specifically binding material and then a 50 mL affinity column at 4 °C, followed by extensive column washing to remove unbound protein.

    Article Title: Monitoring Phospholipase A2 Activity with Gd-encapsulated Phospholipid Liposomes
    Article Snippet: .. Nonentrapped compound was removed via centrifugal filter devices (Amicon Ultra-4, 50 000 MWCO) and size exclusion chromatography using Sepharose CL-4B (Sigma-Aldrich). .. Leakage Assay Measurements of the PLA2 induced release of CF trapped within the liposomes were carried out as follows: CF-loaded liposomal suspensions were incubated in 0.1 Tris (pH 7.4) buffer.

    Protein Binding:

    Article Title: Norbin Stimulates the Catalytic Activity and Plasma Membrane Localization of the Guanine-Nucleotide Exchange Factor P-Rex1 *
    Article Snippet: .. The next day, fractions were pre-cleared for 1 h with 100 μl of Sepharose CL-4B (Sigma) to minimize nonspecific protein binding to beads later on. .. Each cytosol fraction was then split in two, and one-half incubated with 10 μg of purified recombinant human full-length EE-P Rex1 protein that had been immobilized using EE antibody covalently coupled to protein G-Sepharose , the other half with EE-antibody beads alone (without EE-P-Rex1), as a control.

    Article Title: Sec24C/D-isoform–specific sorting of the preassembled ER–Golgi Q-SNARE complex
    Article Snippet: SNARE-GST binding assay For the GST binding assay, 50% (vol/vol) slurry of glutathione Sepharose 4 fast was mixed 1:1 with 50% (vol/vol) slurry of Sepharose CL-4B (Sigma-Aldrich). .. GST or SNARE-GST fusion proteins (100–300 μg, to obtain equal amounts of fusion protein binding) were incubated with 20 μl of the 50% (vol/vol) mixed slurry in assay buffer (25 mM HEPES, pH 7.2 [KOH], 150 mM KOAc, 2 mM MgOAc, 0.02% [vol/vol] monothioglycerol) in a total volume of 1 ml for 1 h at 4°C.

    Produced:

    Article Title: Preparation of Benzothiazolyl-Decorated Nanoliposomes
    Article Snippet: Cholesterol (Chol), SephadexG50 (course), calcein, Sepharose CL-4B were purchased from Sigma-Aldrich OM, Athens, Greece. .. Ultrapure water was produced with a Millipore Direct-Q® 3 with pump system (Millipore S.A., Molsheim, France).

    Article Title: Production, characterization, and biological evaluation of well-defined IgG1 Fc glycoforms as a model system for biosimilarity analysis
    Article Snippet: .. Protein G resin was produced by coupling protein G (recombinantly expressed in E. coli) with Sepharose® CL-4B (Sigma-Aldrich, St Louis, MO) using divinyl sulfone as a coupling reagent. ..

    Immunoprecipitation:

    Article Title: SLAB51 Probiotic Formulation Activates SIRT1 Pathway Promoting Antioxidant and Neuroprotective Effects in an AD Mouse Model
    Article Snippet: Analysis of RARβ Acetylation RARβ was immunoprecipitated from brain homogenates, and acetylated lysines were immunodetected. .. In detail, aliquots of brain homogenates were incubated with agarose-conjugated protein A immobilized on Sepharose CL-4B (Sigma-Aldrich S.r.L., Milano, Italy), followed by SDS-PAGE and immunoblotting.

    Article Title: Proto-oncogene Activity of Melanoma Antigen-A11 (MAGE-A11) Regulates Retinoblastoma-related p107 and E2F1 Proteins *
    Article Snippet: .. Samples were precleared for 15 min at 4 °C using Sepharose CL-4B (Sigma) and immunoprecipitated for 2 h at 4 °C using anti-FLAG M2-agarose affinity resin ( ). .. For phosphorylation experiments, 30 μg of cell extract protein in 40 μl of immunoprecipitation buffer without NaF, sodium vanadate, EDTA, or deoxycholate was incubated for 1 h at 4 °C with or without 4000 IU λ protein phosphatase (New England Biolabs), according to the instructions of the manufacturer ( ).

    Lysis:

    Article Title: Klf5 regulates muscle differentiation by directly targeting muscle-specific genes in cooperation with MyoD in mice
    Article Snippet: After washing with PBS, the cell pellet was lysed in ice-cold Cell Lysis Buffer, incubated on ice for 10–20 min and centrifuged at 3000 rpm for 5 min at 4°C. .. The chromatin was then pre-cleared by incubating with Sepharose CL-4B (Sigma- Aldrich) for 2 hr at 4°C with constant rotation.

    Article Title: Proto-oncogene Activity of Melanoma Antigen-A11 (MAGE-A11) Regulates Retinoblastoma-related p107 and E2F1 Proteins *
    Article Snippet: Samples containing deoxycholate were diluted 4-fold with lysis buffer without deoxycholate. .. Samples were precleared for 15 min at 4 °C using Sepharose CL-4B (Sigma) and immunoprecipitated for 2 h at 4 °C using anti-FLAG M2-agarose affinity resin ( ).

    Staining:

    Article Title: Galectin-3 induces cell migration via a calcium-sensitive MAPK/ERK1/2 pathway
    Article Snippet: Gelcode blue staining regent (24590) and Bio-Rad protein assay kit was both purchased from Bio-Rad Laboratories (Hercules, CA). .. The lactosyl-sepharose CL 4B was prepared using the lactose and sepharose CL 4B (Sigma) according the protocol published previously ( ).

    Article Title: Norbin Stimulates the Catalytic Activity and Plasma Membrane Localization of the Guanine-Nucleotide Exchange Factor P-Rex1 *
    Article Snippet: The next day, fractions were pre-cleared for 1 h with 100 μl of Sepharose CL-4B (Sigma) to minimize nonspecific protein binding to beads later on. .. Protein was denatured in boiling Laemmli sample buffer, resolved on 8% SDS-PAGE gels, and stained using the SilverQuestTM silver staining kit (Invitrogen).

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