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    Structured Review

    Qiagen cell lysates
    Cell Lysates, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cell lysates/product/Qiagen
    Average 99 stars, based on 58 article reviews
    Price from $9.99 to $1999.99
    cell lysates - by Bioz Stars, 2020-03
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    Related Articles

    Clone Assay:

    Article Title: Catalytic-Site Mutations in the MYST Family Histone Acetyltransferase Esa1
    Article Snippet: HIS 6 -ESA1 , HIS 6 -esa1-E338Q , HIS 6 -esa1-C304S , and HIS 6 -esa1-C304S,E338Q were cloned into the pET-15b vector (Novagen, San Diego) and expressed in Escherichia coli strain BL21-CodonPlus(DE3)-RIL cells (Stratagene, La Jolla, CA) by inducing expression with 1 m m IPTG at 28° for 2 hr. .. After centrifugation and flash freezing of cell pellets in a dry ice/ethanol bath, cell lysates were prepared under native conditions and incubated with Ni-NTA beads (QIAGEN, Valencia, CA; cat. no. 1018244) essentially according to the manufacturer's recommended protocol with the following modifications: lysis buffer was 50 m m NaH2 PO4 , 500 m m NaCl, 10 m m imidazole, 10% glycerol, 1% Triton X-100, 10 m m β-mercaptoethanol, pH 8.0; His6 -Esa1 was eluted in 100 μl fractions, and the peak His6 -Esa1 fraction (determined by SDS–PAGE and Coomassie staining) was used as described below; and all steps were performed at room temperature due to aggregation of Esa1 at 4°.

    Centrifugation:

    Article Title: Effects of microvirin monomers and oligomers on hepatitis C virus
    Article Snippet: After 3 h of continuous shaking at 25°C, cells were harvested by centrifugation at 5000 g for 15 min, resuspended in a lysis buffer (50 mM Tris, 100 mM NaCl, 1% Triton X-100, pH 8.0) and disintegrated by sonication (40 on/off cycles with 20 μm amplitude for 15 s at 4°C). .. The resulting cell lysates were centrifuged at 20000 g for 30 min and the supernatants were loaded onto Ni2+ -nitrilotriacetate affinity chromatography columns (Ni-NTA) (Qiagen, 2 ml bed volume) equilibrated with washing buffer (50 mM Tris, 50 mM NaCl, pH 8.0).

    Article Title: Catalytic-Site Mutations in the MYST Family Histone Acetyltransferase Esa1
    Article Snippet: .. After centrifugation and flash freezing of cell pellets in a dry ice/ethanol bath, cell lysates were prepared under native conditions and incubated with Ni-NTA beads (QIAGEN, Valencia, CA; cat. no. 1018244) essentially according to the manufacturer's recommended protocol with the following modifications: lysis buffer was 50 m m NaH2 PO4 , 500 m m NaCl, 10 m m imidazole, 10% glycerol, 1% Triton X-100, 10 m m β-mercaptoethanol, pH 8.0; His6 -Esa1 was eluted in 100 μl fractions, and the peak His6 -Esa1 fraction (determined by SDS–PAGE and Coomassie staining) was used as described below; and all steps were performed at room temperature due to aggregation of Esa1 at 4°. .. His6 -Esa1 (2 μg) was then incubated with chicken core histones and [3 H]-acetyl CoA for 30 min at room temperature followed by separation by SDS–PAGE and fluorography to determine incorporation of [3 H]-acetyl groups into histones.

    Article Title: Epitope Mapping of Function-blocking Monoclonal Antibody CM6 Suggests a "Weak" Integrin Binding Site on the Laminin-332 LG2 Domain
    Article Snippet: After protein induction for 5 h at 34°C, cells were harvested by centrifugation at 6000 rpm for 10 min (Sorvall, GSA rotor). .. Clarified cell lysates were applied to Ni-NTA columns (Qiagen, Valencia, CA).

    Article Title: Roles of the Linker Region of RNA Helicase A in HIV-1 RNA Metabolism
    Article Snippet: .. The cell lysates were cleared by centrifugation and then incubated with Ni-nitrilotriacetic acid (NTA) agarose (Qiagen) at 4°C for 2 hours to capture His-tagged proteins. .. After extensive washing, recombinant proteins were eluted by 250 mM imidazole solution (pH 7.4).

    Article Title: Tropomyosin isoforms differentially tune actin filament length and disassembly
    Article Snippet: .. After a 30 min incubation on ice, cell lysates were cleared by centrifugation at 20,000 × g at 4°C using an eppendorf tabletop centrifuge and incubated with Ni2+ -NTA beads (Qiagen, Valencia, CA) for 90 min at 4°C in the presence of 20 mM imidazole. .. After being washed with Buffer A (20 mM Tris, pH 7.5, 300 mM NaCl, 50 mM imidazole, and 1 mM DTT), proteins were eluted in Buffer A supplemented with 250 mM imidazole, concentrated, and purified further on a Superose 6 gel-filtration column (GE Healthcare Biosciences, Pittsburgh, PA) equilibrated in Buffer B (20 mM Tris, pH 8.0, 50 mM KCl, and 1 mM DTT).

    Amplification:

    Article Title: TSC22D4 is a molecular output of hepatic wasting metabolism
    Article Snippet: .. Quantitative Taqman RT-PCR Total RNA was extracted from homogenized mouse liver or cell lysates using Qiazol reagent (Qiagen, Hilden, Germany). cDNA was prepared by reverse transcription using the M-MuLV enzyme and Oligo dT primer (Fermentas, St. Leon-Rot, Germany). cDNAs were amplified using assay-on-demand kits and an ABIPRISM 7700 Sequence detector (Applied Biosystems, Darmstadt, Germany). .. RNA expression data was normalized to levels of TATA-box binding protein (TBP) RNA.

    Whole Genome Amplification:

    Article Title: Preimplantation Genetic Testing for Monogenic Disease of Spinal Muscular Atrophy by Multiple Displacement Amplification: 11 unaffected livebirths
    Article Snippet: 2.6 Cell lysates and MDA protocol Cell lysis and MDA were performed using the reagents and protocol provided by the manufacturer of the Repli-g Midi kit (Qiagen, Germany). .. The obtained solution (10 μL) was used directly for whole genome amplification (WGA) by adding 40 μL of reaction master mix provided in the kit, followed by incubation at 30 ºC for 8 h and subsequent heat inactivation at 65 ºC for 3 min.

    Synthesized:

    Article Title: ATP-Mediated Transactivation of the Epidermal Growth Factor Receptor in Airway Epithelial Cells Involves DUOX1-Dependent Oxidation of Src and ADAM17
    Article Snippet: .. RT-PCR Analysis Total RNA was extracted from cell lysates using the RNAeasy extraction kit according to the manufacturers protocol (Qiagen, Inc, Valencia, CA) and cDNA was synthesized using M-MLV reverse transcriptase and Oligo(dT)12–16 primer (Invitrogen). .. PCR amplifications were carried out for 30 cycles of denaturation (94°C, 1 min) annealing (58°C, 30 s), and extension (72°C, 1 min), using the following primer sets for DUOX1 ( 5′-GCA GGA CAT CAA CCC TGC ACT CTC-3′ and 5′-CTG CCA TCT ACC ACA CGG ATC TGC-3′ ) and DUOX2 (5′-GAT GGT GAC CGC TAC TGG TT-3′ and 5′-GCC ACC ACT CCA GAG AGA AG-3′), and the products were analyzed on 1% agarose gels.

    Blocking Assay:

    Article Title: Human genome-wide measurement of drug-responsive regulatory activity
    Article Snippet: Reporter output library construction Total RNA was recovered from cell lysates with the Qiagen RNeasy Midi Kit including the on-column DNase I digestion step. .. Eluates were treated with 1 μL RNase Block (Aligent) prior to poly-A RNA isolation with Dynabead Oligo-dT25 beads (Thermo Fisher Scientific) according to the manufacturet’s recommended protocol.

    Enzyme-linked Immunosorbent Assay:

    Article Title: Leptin Locally Synthesized in Carotid Atherosclerotic Plaques Could Be Associated With Lesion Instability and Cerebral Emboli
    Article Snippet: Conditioned media were collected and assayed for leptin antigen level using a Human Leptin ELISA kit (Ray Biotech, Inc, Norcross, GA, USA) according to manufacturer's instructions, where the minimum detectable dose of leptin is typically < 6 pg/mL. .. Cell lysates from human aortic SMC cultures were used to isolate total RNA with an RNeasy kit (Qiagen, Hilden, Germany) and were reverse-transcripted by Superscript II (Invitrogen).

    Microarray:

    Article Title: Human Th17 Cells Lack HIV-Inhibitory RNases and Are Highly Permissive to Productive HIV Infection
    Article Snippet: Paragraph title: RNA purification and microarray. ... Total RNA was extracted from cell lysates using Qiagen Quickspin columns and DNase treatment, according to the manufacturer's protocol (Qiagen Inc., Valencia, CA).

    Article Title: mTORC1/2 inhibition re-sensitizes platinum-resistant ovarian cancer by disrupting selective translation of DNA damage and survival mRNAs
    Article Snippet: For normalization, an aliquot of total RNA was extracted from the same cell lysates used for polysome sedimentation and purified using the QIAGEN protocol. .. 10 µg of pooled RNA was used for microarray analysis using GeneChip Human Gene 2.0 ST Array (Affymetrix), according to the manufacturer instructions.

    Incubation:

    Article Title: Catalytic-Site Mutations in the MYST Family Histone Acetyltransferase Esa1
    Article Snippet: .. After centrifugation and flash freezing of cell pellets in a dry ice/ethanol bath, cell lysates were prepared under native conditions and incubated with Ni-NTA beads (QIAGEN, Valencia, CA; cat. no. 1018244) essentially according to the manufacturer's recommended protocol with the following modifications: lysis buffer was 50 m m NaH2 PO4 , 500 m m NaCl, 10 m m imidazole, 10% glycerol, 1% Triton X-100, 10 m m β-mercaptoethanol, pH 8.0; His6 -Esa1 was eluted in 100 μl fractions, and the peak His6 -Esa1 fraction (determined by SDS–PAGE and Coomassie staining) was used as described below; and all steps were performed at room temperature due to aggregation of Esa1 at 4°. .. His6 -Esa1 (2 μg) was then incubated with chicken core histones and [3 H]-acetyl CoA for 30 min at room temperature followed by separation by SDS–PAGE and fluorography to determine incorporation of [3 H]-acetyl groups into histones.

    Article Title: Preimplantation Genetic Testing for Monogenic Disease of Spinal Muscular Atrophy by Multiple Displacement Amplification: 11 unaffected livebirths
    Article Snippet: 2.6 Cell lysates and MDA protocol Cell lysis and MDA were performed using the reagents and protocol provided by the manufacturer of the Repli-g Midi kit (Qiagen, Germany). .. Samples were mixed with an additional 3.5 μL of freshly prepared lysis buffer and incubated for 10 min at 65 ºC, followed by addition of 3.5 μL of stop buffer.

    Article Title: Roles of the Linker Region of RNA Helicase A in HIV-1 RNA Metabolism
    Article Snippet: .. The cell lysates were cleared by centrifugation and then incubated with Ni-nitrilotriacetic acid (NTA) agarose (Qiagen) at 4°C for 2 hours to capture His-tagged proteins. .. After extensive washing, recombinant proteins were eluted by 250 mM imidazole solution (pH 7.4).

    Article Title: Tropomyosin isoforms differentially tune actin filament length and disassembly
    Article Snippet: .. After a 30 min incubation on ice, cell lysates were cleared by centrifugation at 20,000 × g at 4°C using an eppendorf tabletop centrifuge and incubated with Ni2+ -NTA beads (Qiagen, Valencia, CA) for 90 min at 4°C in the presence of 20 mM imidazole. .. After being washed with Buffer A (20 mM Tris, pH 7.5, 300 mM NaCl, 50 mM imidazole, and 1 mM DTT), proteins were eluted in Buffer A supplemented with 250 mM imidazole, concentrated, and purified further on a Superose 6 gel-filtration column (GE Healthcare Biosciences, Pittsburgh, PA) equilibrated in Buffer B (20 mM Tris, pH 8.0, 50 mM KCl, and 1 mM DTT).

    HAT Assay:

    Article Title: Catalytic-Site Mutations in the MYST Family Histone Acetyltransferase Esa1
    Article Snippet: Paragraph title: Preparation of recombinant Esa1 and HAT assays: ... After centrifugation and flash freezing of cell pellets in a dry ice/ethanol bath, cell lysates were prepared under native conditions and incubated with Ni-NTA beads (QIAGEN, Valencia, CA; cat. no. 1018244) essentially according to the manufacturer's recommended protocol with the following modifications: lysis buffer was 50 m m NaH2 PO4 , 500 m m NaCl, 10 m m imidazole, 10% glycerol, 1% Triton X-100, 10 m m β-mercaptoethanol, pH 8.0; His6 -Esa1 was eluted in 100 μl fractions, and the peak His6 -Esa1 fraction (determined by SDS–PAGE and Coomassie staining) was used as described below; and all steps were performed at room temperature due to aggregation of Esa1 at 4°.

    Expressing:

    Article Title: Human Th17 Cells Lack HIV-Inhibitory RNases and Are Highly Permissive to Productive HIV Infection
    Article Snippet: Total RNA was extracted from cell lysates using Qiagen Quickspin columns and DNase treatment, according to the manufacturer's protocol (Qiagen Inc., Valencia, CA). .. Normalized gene expression estimates were obtained with the Frozen Robust Multiarray Analysis (fRMA) method ( ).

    Article Title: Effects of microvirin monomers and oligomers on hepatitis C virus
    Article Snippet: Paragraph title: Expression and purification of the recombinant MVN oligomers ... The resulting cell lysates were centrifuged at 20000 g for 30 min and the supernatants were loaded onto Ni2+ -nitrilotriacetate affinity chromatography columns (Ni-NTA) (Qiagen, 2 ml bed volume) equilibrated with washing buffer (50 mM Tris, 50 mM NaCl, pH 8.0).

    Article Title: Catalytic-Site Mutations in the MYST Family Histone Acetyltransferase Esa1
    Article Snippet: HIS 6 -ESA1 , HIS 6 -esa1-E338Q , HIS 6 -esa1-C304S , and HIS 6 -esa1-C304S,E338Q were cloned into the pET-15b vector (Novagen, San Diego) and expressed in Escherichia coli strain BL21-CodonPlus(DE3)-RIL cells (Stratagene, La Jolla, CA) by inducing expression with 1 m m IPTG at 28° for 2 hr. .. After centrifugation and flash freezing of cell pellets in a dry ice/ethanol bath, cell lysates were prepared under native conditions and incubated with Ni-NTA beads (QIAGEN, Valencia, CA; cat. no. 1018244) essentially according to the manufacturer's recommended protocol with the following modifications: lysis buffer was 50 m m NaH2 PO4 , 500 m m NaCl, 10 m m imidazole, 10% glycerol, 1% Triton X-100, 10 m m β-mercaptoethanol, pH 8.0; His6 -Esa1 was eluted in 100 μl fractions, and the peak His6 -Esa1 fraction (determined by SDS–PAGE and Coomassie staining) was used as described below; and all steps were performed at room temperature due to aggregation of Esa1 at 4°.

    Article Title: Epitope Mapping of Function-blocking Monoclonal Antibody CM6 Suggests a "Weak" Integrin Binding Site on the Laminin-332 LG2 Domain
    Article Snippet: Paragraph title: Construction, expression, and purification of Ln-332 α3 LG modules ... Clarified cell lysates were applied to Ni-NTA columns (Qiagen, Valencia, CA).

    Article Title: ATP-Mediated Transactivation of the Epidermal Growth Factor Receptor in Airway Epithelial Cells Involves DUOX1-Dependent Oxidation of Src and ADAM17
    Article Snippet: RT-PCR Analysis Total RNA was extracted from cell lysates using the RNAeasy extraction kit according to the manufacturers protocol (Qiagen, Inc, Valencia, CA) and cDNA was synthesized using M-MLV reverse transcriptase and Oligo(dT)12–16 primer (Invitrogen). .. Expression of genes of interest was also analyzed by qPCR relative to GAPDH using SYBR Green PCR Supermix (BioRad) and the following with appropriate primers ( ) and normalized to GAPDH using the ΔΔCT method.

    Article Title: Mesenchymal Stromal Cell-Derived Extracellular Vesicles Attenuate Dendritic Cell Maturation and Function
    Article Snippet: Total RNA from the parent MSCs was purified from cell lysates using the Qiagen RNAeasy mini kit, as per the manufacturer's instructions. .. At least 2 ng of total RNA were used as input for the nCounter® Human miRNA Expression assay V3 kit (NanoString Technologies).

    Article Title: Roles of the Linker Region of RNA Helicase A in HIV-1 RNA Metabolism
    Article Snippet: Paragraph title: Expression and purification of proteins ... The cell lysates were cleared by centrifugation and then incubated with Ni-nitrilotriacetic acid (NTA) agarose (Qiagen) at 4°C for 2 hours to capture His-tagged proteins.

    Genome Wide:

    Article Title: mTORC1/2 inhibition re-sensitizes platinum-resistant ovarian cancer by disrupting selective translation of DNA damage and survival mRNAs
    Article Snippet: Paragraph title: Genome-wide transcription and translation studies ... For normalization, an aliquot of total RNA was extracted from the same cell lysates used for polysome sedimentation and purified using the QIAGEN protocol.

    Modification:

    Article Title: Leptin Locally Synthesized in Carotid Atherosclerotic Plaques Could Be Associated With Lesion Instability and Cerebral Emboli
    Article Snippet: When cells reached 90% confluence, they were preincubated in Dulbecco's modified Eagle medium containing 1% fetal calf serum for 24 hours. .. Cell lysates from human aortic SMC cultures were used to isolate total RNA with an RNeasy kit (Qiagen, Hilden, Germany) and were reverse-transcripted by Superscript II (Invitrogen).

    Transformation Assay:

    Article Title: Effects of microvirin monomers and oligomers on hepatitis C virus
    Article Snippet: Expression and purification of the recombinant MVN oligomers Plasmids containing different MVN variants were transformed into E. coli BL21 (DE3) competent cells, and transformants were grown in 400 ml of fresh LB medium under continuous shaking (250 rpm) at 37°C until OD600 values of cells reached 0.5–0.6. .. The resulting cell lysates were centrifuged at 20000 g for 30 min and the supernatants were loaded onto Ni2+ -nitrilotriacetate affinity chromatography columns (Ni-NTA) (Qiagen, 2 ml bed volume) equilibrated with washing buffer (50 mM Tris, 50 mM NaCl, pH 8.0).

    Activated Clotting Time Assay:

    Article Title: Leptin Locally Synthesized in Carotid Atherosclerotic Plaques Could Be Associated With Lesion Instability and Cerebral Emboli
    Article Snippet: Cell lysates from human aortic SMC cultures were used to isolate total RNA with an RNeasy kit (Qiagen, Hilden, Germany) and were reverse-transcripted by Superscript II (Invitrogen). .. The primer sequences were: leptin, A2074 AA GAG GTT TGG GGT CT, and C2134 CC ACT GTG TGA CAA AAA C; 18S, 5′-ATGGCCGTTCTTAGTTGGTG-3′ and 5′-GAACGCCACTTGTCCCTCTA-3′.

    Article Title: ATP-Mediated Transactivation of the Epidermal Growth Factor Receptor in Airway Epithelial Cells Involves DUOX1-Dependent Oxidation of Src and ADAM17
    Article Snippet: RT-PCR Analysis Total RNA was extracted from cell lysates using the RNAeasy extraction kit according to the manufacturers protocol (Qiagen, Inc, Valencia, CA) and cDNA was synthesized using M-MLV reverse transcriptase and Oligo(dT)12–16 primer (Invitrogen). .. PCR amplifications were carried out for 30 cycles of denaturation (94°C, 1 min) annealing (58°C, 30 s), and extension (72°C, 1 min), using the following primer sets for DUOX1 ( 5′-GCA GGA CAT CAA CCC TGC ACT CTC-3′ and 5′-CTG CCA TCT ACC ACA CGG ATC TGC-3′ ) and DUOX2 (5′-GAT GGT GAC CGC TAC TGG TT-3′ and 5′-GCC ACC ACT CCA GAG AGA AG-3′), and the products were analyzed on 1% agarose gels.

    Derivative Assay:

    Article Title: Human Th17 Cells Lack HIV-Inhibitory RNases and Are Highly Permissive to Productive HIV Infection
    Article Snippet: Total RNA was extracted from cell lysates using Qiagen Quickspin columns and DNase treatment, according to the manufacturer's protocol (Qiagen Inc., Valencia, CA). .. A generalized linear model approach, coupled with empirical Bayes standard error shrinkage and including coefficients for data heterogeneity as derived from surrogate variable analysis (SVA) , was used for identifying differentially expressed genes in Th 17-polarized cells relative to Th 0-polarized cells.

    Countercurrent Chromatography:

    Article Title: ATP-Mediated Transactivation of the Epidermal Growth Factor Receptor in Airway Epithelial Cells Involves DUOX1-Dependent Oxidation of Src and ADAM17
    Article Snippet: RT-PCR Analysis Total RNA was extracted from cell lysates using the RNAeasy extraction kit according to the manufacturers protocol (Qiagen, Inc, Valencia, CA) and cDNA was synthesized using M-MLV reverse transcriptase and Oligo(dT)12–16 primer (Invitrogen). .. PCR amplifications were carried out for 30 cycles of denaturation (94°C, 1 min) annealing (58°C, 30 s), and extension (72°C, 1 min), using the following primer sets for DUOX1 ( 5′-GCA GGA CAT CAA CCC TGC ACT CTC-3′ and 5′-CTG CCA TCT ACC ACA CGG ATC TGC-3′ ) and DUOX2 (5′-GAT GGT GAC CGC TAC TGG TT-3′ and 5′-GCC ACC ACT CCA GAG AGA AG-3′), and the products were analyzed on 1% agarose gels.

    Transfection:

    Article Title: Roles of the Linker Region of RNA Helicase A in HIV-1 RNA Metabolism
    Article Snippet: Briefly, cells were transfected with indicated plasmids using 25 kDa linear Polyethylenimine (PEI, pH7.0, Polysciences Inc.), collected 48 hours later, washed with ice-cold phosphate-buffered saline, and lysed in lysis buffer [50 mM NaH2 PO4 , pH 7.4, 300 mM NaCl, 10 mM imidazole, 0.5% Triton-X 100, 10% glycerol, and protease inhibitor cocktail tablets (Roche)]. .. The cell lysates were cleared by centrifugation and then incubated with Ni-nitrilotriacetic acid (NTA) agarose (Qiagen) at 4°C for 2 hours to capture His-tagged proteins.

    Article Title: Tropomyosin isoforms differentially tune actin filament length and disassembly
    Article Snippet: Cells at 30–40% confluence were transiently transfected using 25 kDa linear polyethylenimine (Polysciences, Warrington, PA). .. After a 30 min incubation on ice, cell lysates were cleared by centrifugation at 20,000 × g at 4°C using an eppendorf tabletop centrifuge and incubated with Ni2+ -NTA beads (Qiagen, Valencia, CA) for 90 min at 4°C in the presence of 20 mM imidazole.

    Sequencing:

    Article Title: TSC22D4 is a molecular output of hepatic wasting metabolism
    Article Snippet: .. Quantitative Taqman RT-PCR Total RNA was extracted from homogenized mouse liver or cell lysates using Qiazol reagent (Qiagen, Hilden, Germany). cDNA was prepared by reverse transcription using the M-MuLV enzyme and Oligo dT primer (Fermentas, St. Leon-Rot, Germany). cDNAs were amplified using assay-on-demand kits and an ABIPRISM 7700 Sequence detector (Applied Biosystems, Darmstadt, Germany). .. RNA expression data was normalized to levels of TATA-box binding protein (TBP) RNA.

    Chromatography:

    Article Title: Tropomyosin isoforms differentially tune actin filament length and disassembly
    Article Snippet: The formin mDia1 (6xHis-FH1-FH2-C) was inducibly expressed in yeast and purified by sequential Ni2+ -NTA and gel-filtration chromatography steps. .. After a 30 min incubation on ice, cell lysates were cleared by centrifugation at 20,000 × g at 4°C using an eppendorf tabletop centrifuge and incubated with Ni2+ -NTA beads (Qiagen, Valencia, CA) for 90 min at 4°C in the presence of 20 mM imidazole.

    Protease Inhibitor:

    Article Title: Roles of the Linker Region of RNA Helicase A in HIV-1 RNA Metabolism
    Article Snippet: Briefly, cells were transfected with indicated plasmids using 25 kDa linear Polyethylenimine (PEI, pH7.0, Polysciences Inc.), collected 48 hours later, washed with ice-cold phosphate-buffered saline, and lysed in lysis buffer [50 mM NaH2 PO4 , pH 7.4, 300 mM NaCl, 10 mM imidazole, 0.5% Triton-X 100, 10% glycerol, and protease inhibitor cocktail tablets (Roche)]. .. The cell lysates were cleared by centrifugation and then incubated with Ni-nitrilotriacetic acid (NTA) agarose (Qiagen) at 4°C for 2 hours to capture His-tagged proteins.

    Cell Culture:

    Article Title: Effects of microvirin monomers and oligomers on hepatitis C virus
    Article Snippet: To induce the expression of recombinant proteins, isopropyl β-D -thiogalactopyranoside (IPTG) at a final concentration of 1 mM was added to the cell culture. .. The resulting cell lysates were centrifuged at 20000 g for 30 min and the supernatants were loaded onto Ni2+ -nitrilotriacetate affinity chromatography columns (Ni-NTA) (Qiagen, 2 ml bed volume) equilibrated with washing buffer (50 mM Tris, 50 mM NaCl, pH 8.0).

    Article Title: Leptin Locally Synthesized in Carotid Atherosclerotic Plaques Could Be Associated With Lesion Instability and Cerebral Emboli
    Article Snippet: Cells were cultured in Dulbecco's modified Eagle medium containing 10% fetal calf serum and were used between passages 3 and 6. .. Cell lysates from human aortic SMC cultures were used to isolate total RNA with an RNeasy kit (Qiagen, Hilden, Germany) and were reverse-transcripted by Superscript II (Invitrogen).

    Sedimentation:

    Article Title: mTORC1/2 inhibition re-sensitizes platinum-resistant ovarian cancer by disrupting selective translation of DNA damage and survival mRNAs
    Article Snippet: .. For normalization, an aliquot of total RNA was extracted from the same cell lysates used for polysome sedimentation and purified using the QIAGEN protocol. .. RNA quantity and quality for all samples were determined by bioanalysis (Agilent Technologies), and samples stored in nuclease-free water at −80°C.

    Polymerase Chain Reaction:

    Article Title: ATP-Mediated Transactivation of the Epidermal Growth Factor Receptor in Airway Epithelial Cells Involves DUOX1-Dependent Oxidation of Src and ADAM17
    Article Snippet: RT-PCR Analysis Total RNA was extracted from cell lysates using the RNAeasy extraction kit according to the manufacturers protocol (Qiagen, Inc, Valencia, CA) and cDNA was synthesized using M-MLV reverse transcriptase and Oligo(dT)12–16 primer (Invitrogen). .. PCR amplifications were carried out for 30 cycles of denaturation (94°C, 1 min) annealing (58°C, 30 s), and extension (72°C, 1 min), using the following primer sets for DUOX1 ( 5′-GCA GGA CAT CAA CCC TGC ACT CTC-3′ and 5′-CTG CCA TCT ACC ACA CGG ATC TGC-3′ ) and DUOX2 (5′-GAT GGT GAC CGC TAC TGG TT-3′ and 5′-GCC ACC ACT CCA GAG AGA AG-3′), and the products were analyzed on 1% agarose gels.

    Article Title: Involvement in surface antigen expression by a moonlighting FG-repeat nucleoporin in trypanosomes
    Article Snippet: .. Real-time quantitative reverse transcriptase PCR RNA was purified from cell lysates using an RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. .. Superscript III Reverse Transcriptase (Invitrogen) accompanied with RNase OUT (Invitrogen) was used to synthesize cDNA from 1 μg RNA. qRT–PCR using cDNA templates was carried out using iQ-SYBRGreen Supermix and a MiniOpticon Real-Time PCR Detection System (Bio-Rad).

    Sonication:

    Article Title: Effects of microvirin monomers and oligomers on hepatitis C virus
    Article Snippet: After 3 h of continuous shaking at 25°C, cells were harvested by centrifugation at 5000 g for 15 min, resuspended in a lysis buffer (50 mM Tris, 100 mM NaCl, 1% Triton X-100, pH 8.0) and disintegrated by sonication (40 on/off cycles with 20 μm amplitude for 15 s at 4°C). .. The resulting cell lysates were centrifuged at 20000 g for 30 min and the supernatants were loaded onto Ni2+ -nitrilotriacetate affinity chromatography columns (Ni-NTA) (Qiagen, 2 ml bed volume) equilibrated with washing buffer (50 mM Tris, 50 mM NaCl, pH 8.0).

    Binding Assay:

    Article Title: TSC22D4 is a molecular output of hepatic wasting metabolism
    Article Snippet: Quantitative Taqman RT-PCR Total RNA was extracted from homogenized mouse liver or cell lysates using Qiazol reagent (Qiagen, Hilden, Germany). cDNA was prepared by reverse transcription using the M-MuLV enzyme and Oligo dT primer (Fermentas, St. Leon-Rot, Germany). cDNAs were amplified using assay-on-demand kits and an ABIPRISM 7700 Sequence detector (Applied Biosystems, Darmstadt, Germany). .. RNA expression data was normalized to levels of TATA-box binding protein (TBP) RNA.

    Cellular Antioxidant Activity Assay:

    Article Title: Leptin Locally Synthesized in Carotid Atherosclerotic Plaques Could Be Associated With Lesion Instability and Cerebral Emboli
    Article Snippet: Cell lysates from human aortic SMC cultures were used to isolate total RNA with an RNeasy kit (Qiagen, Hilden, Germany) and were reverse-transcripted by Superscript II (Invitrogen). .. The primer sequences were: leptin, A2074 AA GAG GTT TGG GGT CT, and C2134 CC ACT GTG TGA CAA AAA C; 18S, 5′-ATGGCCGTTCTTAGTTGGTG-3′ and 5′-GAACGCCACTTGTCCCTCTA-3′.

    Article Title: ATP-Mediated Transactivation of the Epidermal Growth Factor Receptor in Airway Epithelial Cells Involves DUOX1-Dependent Oxidation of Src and ADAM17
    Article Snippet: RT-PCR Analysis Total RNA was extracted from cell lysates using the RNAeasy extraction kit according to the manufacturers protocol (Qiagen, Inc, Valencia, CA) and cDNA was synthesized using M-MLV reverse transcriptase and Oligo(dT)12–16 primer (Invitrogen). .. PCR amplifications were carried out for 30 cycles of denaturation (94°C, 1 min) annealing (58°C, 30 s), and extension (72°C, 1 min), using the following primer sets for DUOX1 ( 5′-GCA GGA CAT CAA CCC TGC ACT CTC-3′ and 5′-CTG CCA TCT ACC ACA CGG ATC TGC-3′ ) and DUOX2 (5′-GAT GGT GAC CGC TAC TGG TT-3′ and 5′-GCC ACC ACT CCA GAG AGA AG-3′), and the products were analyzed on 1% agarose gels.

    Real-time Polymerase Chain Reaction:

    Article Title: Leptin Locally Synthesized in Carotid Atherosclerotic Plaques Could Be Associated With Lesion Instability and Cerebral Emboli
    Article Snippet: Cell lysates from human aortic SMC cultures were used to isolate total RNA with an RNeasy kit (Qiagen, Hilden, Germany) and were reverse-transcripted by Superscript II (Invitrogen). .. Quantitative PCR was performed for leptin mRNA in a MyiQ Single-Color Real-Time PCR system with SYBR Green I (Bio-Rad, Hercules, CA, USA).

    Article Title: ATP-Mediated Transactivation of the Epidermal Growth Factor Receptor in Airway Epithelial Cells Involves DUOX1-Dependent Oxidation of Src and ADAM17
    Article Snippet: RT-PCR Analysis Total RNA was extracted from cell lysates using the RNAeasy extraction kit according to the manufacturers protocol (Qiagen, Inc, Valencia, CA) and cDNA was synthesized using M-MLV reverse transcriptase and Oligo(dT)12–16 primer (Invitrogen). .. Expression of genes of interest was also analyzed by qPCR relative to GAPDH using SYBR Green PCR Supermix (BioRad) and the following with appropriate primers ( ) and normalized to GAPDH using the ΔΔCT method.

    Article Title: Involvement in surface antigen expression by a moonlighting FG-repeat nucleoporin in trypanosomes
    Article Snippet: Real-time quantitative reverse transcriptase PCR RNA was purified from cell lysates using an RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. .. Superscript III Reverse Transcriptase (Invitrogen) accompanied with RNase OUT (Invitrogen) was used to synthesize cDNA from 1 μg RNA. qRT–PCR using cDNA templates was carried out using iQ-SYBRGreen Supermix and a MiniOpticon Real-Time PCR Detection System (Bio-Rad).

    Article Title: SerpinA3N is a novel hypothalamic gene upregulated by a high-fat diet and leptin in mice
    Article Snippet: Paragraph title: Real-time PCR ... Cell lysates were then collected and RNA extracted using an RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions.

    Fluorescence:

    Article Title: Tropomyosin isoforms differentially tune actin filament length and disassembly
    Article Snippet: After a 30 min incubation on ice, cell lysates were cleared by centrifugation at 20,000 × g at 4°C using an eppendorf tabletop centrifuge and incubated with Ni2+ -NTA beads (Qiagen, Valencia, CA) for 90 min at 4°C in the presence of 20 mM imidazole. .. For fluorescence labeling of SNAP-Cor1B, the fusion protein was bound to Ni2+ -NTA beads, washed extensively in PBS with 1 mM DTT, and incubated with a fivefold excess of benzylguanine or benzylchloropyrimidine SNAP-Surface 649 (New England Biolabs, Ipswich, MA) for 2 h at room temperature.

    Mutagenesis:

    Article Title: Roles of the Linker Region of RNA Helicase A in HIV-1 RNA Metabolism
    Article Snippet: Expression and purification of proteins The N-terminally 6×His-tagged mutant RHAs containing deletions in the linker region were expressed and isolated as described from HEK 293E cells . .. The cell lysates were cleared by centrifugation and then incubated with Ni-nitrilotriacetic acid (NTA) agarose (Qiagen) at 4°C for 2 hours to capture His-tagged proteins.

    Isolation:

    Article Title: mTORC1/2 inhibition re-sensitizes platinum-resistant ovarian cancer by disrupting selective translation of DNA damage and survival mRNAs
    Article Snippet: RNA was isolated from each fraction by extraction using the QIAGEN RNeasy MinElute Cleanup Kit. .. For normalization, an aliquot of total RNA was extracted from the same cell lysates used for polysome sedimentation and purified using the QIAGEN protocol.

    Article Title: Leptin Locally Synthesized in Carotid Atherosclerotic Plaques Could Be Associated With Lesion Instability and Cerebral Emboli
    Article Snippet: Human venous SMCs were isolated by explantation from saphenous veins obtained for cardiac bypass surgery. .. Cell lysates from human aortic SMC cultures were used to isolate total RNA with an RNeasy kit (Qiagen, Hilden, Germany) and were reverse-transcripted by Superscript II (Invitrogen).

    Article Title: Human genome-wide measurement of drug-responsive regulatory activity
    Article Snippet: Reporter output library construction Total RNA was recovered from cell lysates with the Qiagen RNeasy Midi Kit including the on-column DNase I digestion step. .. Eluates were treated with 1 μL RNase Block (Aligent) prior to poly-A RNA isolation with Dynabead Oligo-dT25 beads (Thermo Fisher Scientific) according to the manufacturet’s recommended protocol.

    Article Title: Mesenchymal Stromal Cell-Derived Extracellular Vesicles Attenuate Dendritic Cell Maturation and Function
    Article Snippet: MSC-EV miRNA profiling Total RNA in MSC-EVs was purified using the Total Exosome RNA and Protein isolation kit (Thermo Fisher Scientific). .. Total RNA from the parent MSCs was purified from cell lysates using the Qiagen RNAeasy mini kit, as per the manufacturer's instructions.

    Article Title: Roles of the Linker Region of RNA Helicase A in HIV-1 RNA Metabolism
    Article Snippet: Expression and purification of proteins The N-terminally 6×His-tagged mutant RHAs containing deletions in the linker region were expressed and isolated as described from HEK 293E cells . .. The cell lysates were cleared by centrifugation and then incubated with Ni-nitrilotriacetic acid (NTA) agarose (Qiagen) at 4°C for 2 hours to capture His-tagged proteins.

    Labeling:

    Article Title: Tropomyosin isoforms differentially tune actin filament length and disassembly
    Article Snippet: Paragraph title: Protein purification and labeling ... After a 30 min incubation on ice, cell lysates were cleared by centrifugation at 20,000 × g at 4°C using an eppendorf tabletop centrifuge and incubated with Ni2+ -NTA beads (Qiagen, Valencia, CA) for 90 min at 4°C in the presence of 20 mM imidazole.

    Purification:

    Article Title: Human Th17 Cells Lack HIV-Inhibitory RNases and Are Highly Permissive to Productive HIV Infection
    Article Snippet: Paragraph title: RNA purification and microarray. ... Total RNA was extracted from cell lysates using Qiagen Quickspin columns and DNase treatment, according to the manufacturer's protocol (Qiagen Inc., Valencia, CA).

    Article Title: Effects of microvirin monomers and oligomers on hepatitis C virus
    Article Snippet: Paragraph title: Expression and purification of the recombinant MVN oligomers ... The resulting cell lysates were centrifuged at 20000 g for 30 min and the supernatants were loaded onto Ni2+ -nitrilotriacetate affinity chromatography columns (Ni-NTA) (Qiagen, 2 ml bed volume) equilibrated with washing buffer (50 mM Tris, 50 mM NaCl, pH 8.0).

    Article Title: mTORC1/2 inhibition re-sensitizes platinum-resistant ovarian cancer by disrupting selective translation of DNA damage and survival mRNAs
    Article Snippet: .. For normalization, an aliquot of total RNA was extracted from the same cell lysates used for polysome sedimentation and purified using the QIAGEN protocol. .. RNA quantity and quality for all samples were determined by bioanalysis (Agilent Technologies), and samples stored in nuclease-free water at −80°C.

    Article Title: Epitope Mapping of Function-blocking Monoclonal Antibody CM6 Suggests a "Weak" Integrin Binding Site on the Laminin-332 LG2 Domain
    Article Snippet: Paragraph title: Construction, expression, and purification of Ln-332 α3 LG modules ... Clarified cell lysates were applied to Ni-NTA columns (Qiagen, Valencia, CA).

    Article Title: Mesenchymal Stromal Cell-Derived Extracellular Vesicles Attenuate Dendritic Cell Maturation and Function
    Article Snippet: .. Total RNA from the parent MSCs was purified from cell lysates using the Qiagen RNAeasy mini kit, as per the manufacturer's instructions. .. The concentration and quality of total RNA from MSC and MSC-EVs was determined using Bioanalyzer 2100 with the RNA 6000 Nano and Pico kits, respectively (Agilent technologies).

    Article Title: Roles of the Linker Region of RNA Helicase A in HIV-1 RNA Metabolism
    Article Snippet: Paragraph title: Expression and purification of proteins ... The cell lysates were cleared by centrifugation and then incubated with Ni-nitrilotriacetic acid (NTA) agarose (Qiagen) at 4°C for 2 hours to capture His-tagged proteins.

    Article Title: Involvement in surface antigen expression by a moonlighting FG-repeat nucleoporin in trypanosomes
    Article Snippet: .. Real-time quantitative reverse transcriptase PCR RNA was purified from cell lysates using an RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. .. Superscript III Reverse Transcriptase (Invitrogen) accompanied with RNase OUT (Invitrogen) was used to synthesize cDNA from 1 μg RNA. qRT–PCR using cDNA templates was carried out using iQ-SYBRGreen Supermix and a MiniOpticon Real-Time PCR Detection System (Bio-Rad).

    Article Title: Tropomyosin isoforms differentially tune actin filament length and disassembly
    Article Snippet: Cor1B and AIP1 were expressed and purified from transfected HEK293T cells (ATCC). .. After a 30 min incubation on ice, cell lysates were cleared by centrifugation at 20,000 × g at 4°C using an eppendorf tabletop centrifuge and incubated with Ni2+ -NTA beads (Qiagen, Valencia, CA) for 90 min at 4°C in the presence of 20 mM imidazole.

    Protein Purification:

    Article Title: Effects of microvirin monomers and oligomers on hepatitis C virus
    Article Snippet: The resulting cell lysates were centrifuged at 20000 g for 30 min and the supernatants were loaded onto Ni2+ -nitrilotriacetate affinity chromatography columns (Ni-NTA) (Qiagen, 2 ml bed volume) equilibrated with washing buffer (50 mM Tris, 50 mM NaCl, pH 8.0). .. The fractions with the highest absorbance at 280 nm (approximately 20–50% of the total protein retained, Supplementary Table S2) were combined and desalted with PD-10 protein purification columns (GE Healthcare, pre-equilibrated with 20 mM piperazine) using 20 mM piperazine as mobile phase.

    Article Title: Epitope Mapping of Function-blocking Monoclonal Antibody CM6 Suggests a "Weak" Integrin Binding Site on the Laminin-332 LG2 Domain
    Article Snippet: For protein purification, pellets were thawed and resuspended in ice-cold lysis buffer (Tris-HCl, 20 mM, ph 7.8; glycerol, 10%; NaCl, 0.15 M; imidazole, 5 mM; Triton-X-100, 1%) containing 1 mM phenylmethylsulphonyl fluoride (PMSF; Sigma). .. Clarified cell lysates were applied to Ni-NTA columns (Qiagen, Valencia, CA).

    Article Title: Tropomyosin isoforms differentially tune actin filament length and disassembly
    Article Snippet: Paragraph title: Protein purification and labeling ... After a 30 min incubation on ice, cell lysates were cleared by centrifugation at 20,000 × g at 4°C using an eppendorf tabletop centrifuge and incubated with Ni2+ -NTA beads (Qiagen, Valencia, CA) for 90 min at 4°C in the presence of 20 mM imidazole.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: TSC22D4 is a molecular output of hepatic wasting metabolism
    Article Snippet: .. Quantitative Taqman RT-PCR Total RNA was extracted from homogenized mouse liver or cell lysates using Qiazol reagent (Qiagen, Hilden, Germany). cDNA was prepared by reverse transcription using the M-MuLV enzyme and Oligo dT primer (Fermentas, St. Leon-Rot, Germany). cDNAs were amplified using assay-on-demand kits and an ABIPRISM 7700 Sequence detector (Applied Biosystems, Darmstadt, Germany). .. RNA expression data was normalized to levels of TATA-box binding protein (TBP) RNA.

    Article Title: ATP-Mediated Transactivation of the Epidermal Growth Factor Receptor in Airway Epithelial Cells Involves DUOX1-Dependent Oxidation of Src and ADAM17
    Article Snippet: .. RT-PCR Analysis Total RNA was extracted from cell lysates using the RNAeasy extraction kit according to the manufacturers protocol (Qiagen, Inc, Valencia, CA) and cDNA was synthesized using M-MLV reverse transcriptase and Oligo(dT)12–16 primer (Invitrogen). .. PCR amplifications were carried out for 30 cycles of denaturation (94°C, 1 min) annealing (58°C, 30 s), and extension (72°C, 1 min), using the following primer sets for DUOX1 ( 5′-GCA GGA CAT CAA CCC TGC ACT CTC-3′ and 5′-CTG CCA TCT ACC ACA CGG ATC TGC-3′ ) and DUOX2 (5′-GAT GGT GAC CGC TAC TGG TT-3′ and 5′-GCC ACC ACT CCA GAG AGA AG-3′), and the products were analyzed on 1% agarose gels.

    Positron Emission Tomography:

    Article Title: Catalytic-Site Mutations in the MYST Family Histone Acetyltransferase Esa1
    Article Snippet: HIS 6 -ESA1 , HIS 6 -esa1-E338Q , HIS 6 -esa1-C304S , and HIS 6 -esa1-C304S,E338Q were cloned into the pET-15b vector (Novagen, San Diego) and expressed in Escherichia coli strain BL21-CodonPlus(DE3)-RIL cells (Stratagene, La Jolla, CA) by inducing expression with 1 m m IPTG at 28° for 2 hr. .. After centrifugation and flash freezing of cell pellets in a dry ice/ethanol bath, cell lysates were prepared under native conditions and incubated with Ni-NTA beads (QIAGEN, Valencia, CA; cat. no. 1018244) essentially according to the manufacturer's recommended protocol with the following modifications: lysis buffer was 50 m m NaH2 PO4 , 500 m m NaCl, 10 m m imidazole, 10% glycerol, 1% Triton X-100, 10 m m β-mercaptoethanol, pH 8.0; His6 -Esa1 was eluted in 100 μl fractions, and the peak His6 -Esa1 fraction (determined by SDS–PAGE and Coomassie staining) was used as described below; and all steps were performed at room temperature due to aggregation of Esa1 at 4°.

    Quantitative RT-PCR:

    Article Title: Involvement in surface antigen expression by a moonlighting FG-repeat nucleoporin in trypanosomes
    Article Snippet: Real-time quantitative reverse transcriptase PCR RNA was purified from cell lysates using an RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. .. Superscript III Reverse Transcriptase (Invitrogen) accompanied with RNase OUT (Invitrogen) was used to synthesize cDNA from 1 μg RNA. qRT–PCR using cDNA templates was carried out using iQ-SYBRGreen Supermix and a MiniOpticon Real-Time PCR Detection System (Bio-Rad).

    Staining:

    Article Title: Catalytic-Site Mutations in the MYST Family Histone Acetyltransferase Esa1
    Article Snippet: .. After centrifugation and flash freezing of cell pellets in a dry ice/ethanol bath, cell lysates were prepared under native conditions and incubated with Ni-NTA beads (QIAGEN, Valencia, CA; cat. no. 1018244) essentially according to the manufacturer's recommended protocol with the following modifications: lysis buffer was 50 m m NaH2 PO4 , 500 m m NaCl, 10 m m imidazole, 10% glycerol, 1% Triton X-100, 10 m m β-mercaptoethanol, pH 8.0; His6 -Esa1 was eluted in 100 μl fractions, and the peak His6 -Esa1 fraction (determined by SDS–PAGE and Coomassie staining) was used as described below; and all steps were performed at room temperature due to aggregation of Esa1 at 4°. .. His6 -Esa1 (2 μg) was then incubated with chicken core histones and [3 H]-acetyl CoA for 30 min at room temperature followed by separation by SDS–PAGE and fluorography to determine incorporation of [3 H]-acetyl groups into histones.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: ATP-Mediated Transactivation of the Epidermal Growth Factor Receptor in Airway Epithelial Cells Involves DUOX1-Dependent Oxidation of Src and ADAM17
    Article Snippet: RT-PCR Analysis Total RNA was extracted from cell lysates using the RNAeasy extraction kit according to the manufacturers protocol (Qiagen, Inc, Valencia, CA) and cDNA was synthesized using M-MLV reverse transcriptase and Oligo(dT)12–16 primer (Invitrogen). .. PCR amplifications were carried out for 30 cycles of denaturation (94°C, 1 min) annealing (58°C, 30 s), and extension (72°C, 1 min), using the following primer sets for DUOX1 ( 5′-GCA GGA CAT CAA CCC TGC ACT CTC-3′ and 5′-CTG CCA TCT ACC ACA CGG ATC TGC-3′ ) and DUOX2 (5′-GAT GGT GAC CGC TAC TGG TT-3′ and 5′-GCC ACC ACT CCA GAG AGA AG-3′), and the products were analyzed on 1% agarose gels.

    SDS Page:

    Article Title: Catalytic-Site Mutations in the MYST Family Histone Acetyltransferase Esa1
    Article Snippet: .. After centrifugation and flash freezing of cell pellets in a dry ice/ethanol bath, cell lysates were prepared under native conditions and incubated with Ni-NTA beads (QIAGEN, Valencia, CA; cat. no. 1018244) essentially according to the manufacturer's recommended protocol with the following modifications: lysis buffer was 50 m m NaH2 PO4 , 500 m m NaCl, 10 m m imidazole, 10% glycerol, 1% Triton X-100, 10 m m β-mercaptoethanol, pH 8.0; His6 -Esa1 was eluted in 100 μl fractions, and the peak His6 -Esa1 fraction (determined by SDS–PAGE and Coomassie staining) was used as described below; and all steps were performed at room temperature due to aggregation of Esa1 at 4°. .. His6 -Esa1 (2 μg) was then incubated with chicken core histones and [3 H]-acetyl CoA for 30 min at room temperature followed by separation by SDS–PAGE and fluorography to determine incorporation of [3 H]-acetyl groups into histones.

    Plasmid Preparation:

    Article Title: Catalytic-Site Mutations in the MYST Family Histone Acetyltransferase Esa1
    Article Snippet: HIS 6 -ESA1 , HIS 6 -esa1-E338Q , HIS 6 -esa1-C304S , and HIS 6 -esa1-C304S,E338Q were cloned into the pET-15b vector (Novagen, San Diego) and expressed in Escherichia coli strain BL21-CodonPlus(DE3)-RIL cells (Stratagene, La Jolla, CA) by inducing expression with 1 m m IPTG at 28° for 2 hr. .. After centrifugation and flash freezing of cell pellets in a dry ice/ethanol bath, cell lysates were prepared under native conditions and incubated with Ni-NTA beads (QIAGEN, Valencia, CA; cat. no. 1018244) essentially according to the manufacturer's recommended protocol with the following modifications: lysis buffer was 50 m m NaH2 PO4 , 500 m m NaCl, 10 m m imidazole, 10% glycerol, 1% Triton X-100, 10 m m β-mercaptoethanol, pH 8.0; His6 -Esa1 was eluted in 100 μl fractions, and the peak His6 -Esa1 fraction (determined by SDS–PAGE and Coomassie staining) was used as described below; and all steps were performed at room temperature due to aggregation of Esa1 at 4°.

    Software:

    Article Title: Mesenchymal Stromal Cell-Derived Extracellular Vesicles Attenuate Dendritic Cell Maturation and Function
    Article Snippet: Total RNA from the parent MSCs was purified from cell lysates using the Qiagen RNAeasy mini kit, as per the manufacturer's instructions. .. The miRNA expression profiles were then analyzed using the nSolver software V2.5 (NanoString Technologies) according to the manufacturer's instructions.

    Article Title: Involvement in surface antigen expression by a moonlighting FG-repeat nucleoporin in trypanosomes
    Article Snippet: Real-time quantitative reverse transcriptase PCR RNA was purified from cell lysates using an RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. .. Results were analyzed using MiniOpticon software (Bio-Rad) with β-tubulin as a housekeeping gene to normalize RNA input.

    SYBR Green Assay:

    Article Title: Leptin Locally Synthesized in Carotid Atherosclerotic Plaques Could Be Associated With Lesion Instability and Cerebral Emboli
    Article Snippet: Cell lysates from human aortic SMC cultures were used to isolate total RNA with an RNeasy kit (Qiagen, Hilden, Germany) and were reverse-transcripted by Superscript II (Invitrogen). .. Quantitative PCR was performed for leptin mRNA in a MyiQ Single-Color Real-Time PCR system with SYBR Green I (Bio-Rad, Hercules, CA, USA).

    Article Title: ATP-Mediated Transactivation of the Epidermal Growth Factor Receptor in Airway Epithelial Cells Involves DUOX1-Dependent Oxidation of Src and ADAM17
    Article Snippet: RT-PCR Analysis Total RNA was extracted from cell lysates using the RNAeasy extraction kit according to the manufacturers protocol (Qiagen, Inc, Valencia, CA) and cDNA was synthesized using M-MLV reverse transcriptase and Oligo(dT)12–16 primer (Invitrogen). .. Expression of genes of interest was also analyzed by qPCR relative to GAPDH using SYBR Green PCR Supermix (BioRad) and the following with appropriate primers ( ) and normalized to GAPDH using the ΔΔCT method.

    RNA Expression:

    Article Title: TSC22D4 is a molecular output of hepatic wasting metabolism
    Article Snippet: Quantitative Taqman RT-PCR Total RNA was extracted from homogenized mouse liver or cell lysates using Qiazol reagent (Qiagen, Hilden, Germany). cDNA was prepared by reverse transcription using the M-MuLV enzyme and Oligo dT primer (Fermentas, St. Leon-Rot, Germany). cDNAs were amplified using assay-on-demand kits and an ABIPRISM 7700 Sequence detector (Applied Biosystems, Darmstadt, Germany). .. RNA expression data was normalized to levels of TATA-box binding protein (TBP) RNA.

    In Vitro:

    Article Title: Leptin Locally Synthesized in Carotid Atherosclerotic Plaques Could Be Associated With Lesion Instability and Cerebral Emboli
    Article Snippet: Paragraph title: In Vitro Studies in Human Aortic and Human Saphenous Vein SMCs ... Cell lysates from human aortic SMC cultures were used to isolate total RNA with an RNeasy kit (Qiagen, Hilden, Germany) and were reverse-transcripted by Superscript II (Invitrogen).

    Affinity Chromatography:

    Article Title: Effects of microvirin monomers and oligomers on hepatitis C virus
    Article Snippet: .. The resulting cell lysates were centrifuged at 20000 g for 30 min and the supernatants were loaded onto Ni2+ -nitrilotriacetate affinity chromatography columns (Ni-NTA) (Qiagen, 2 ml bed volume) equilibrated with washing buffer (50 mM Tris, 50 mM NaCl, pH 8.0). ..

    Concentration Assay:

    Article Title: Effects of microvirin monomers and oligomers on hepatitis C virus
    Article Snippet: To induce the expression of recombinant proteins, isopropyl β-D -thiogalactopyranoside (IPTG) at a final concentration of 1 mM was added to the cell culture. .. The resulting cell lysates were centrifuged at 20000 g for 30 min and the supernatants were loaded onto Ni2+ -nitrilotriacetate affinity chromatography columns (Ni-NTA) (Qiagen, 2 ml bed volume) equilibrated with washing buffer (50 mM Tris, 50 mM NaCl, pH 8.0).

    Article Title: mTORC1/2 inhibition re-sensitizes platinum-resistant ovarian cancer by disrupting selective translation of DNA damage and survival mRNAs
    Article Snippet: RNA concentration of each sample was determined by NanoDrop and equal RNA amounts (400 µg/mL) layered onto 15-50% sterile sucrose gradients in polysome extraction buffer supplemented with 100 µg/mL cycloheximide. .. For normalization, an aliquot of total RNA was extracted from the same cell lysates used for polysome sedimentation and purified using the QIAGEN protocol.

    Article Title: Mesenchymal Stromal Cell-Derived Extracellular Vesicles Attenuate Dendritic Cell Maturation and Function
    Article Snippet: Total RNA from the parent MSCs was purified from cell lysates using the Qiagen RNAeasy mini kit, as per the manufacturer's instructions. .. The concentration and quality of total RNA from MSC and MSC-EVs was determined using Bioanalyzer 2100 with the RNA 6000 Nano and Pico kits, respectively (Agilent technologies).

    Multiple Displacement Amplification:

    Article Title: Preimplantation Genetic Testing for Monogenic Disease of Spinal Muscular Atrophy by Multiple Displacement Amplification: 11 unaffected livebirths
    Article Snippet: .. 2.6 Cell lysates and MDA protocol Cell lysis and MDA were performed using the reagents and protocol provided by the manufacturer of the Repli-g Midi kit (Qiagen, Germany). .. Samples were mixed with an additional 3.5 μL of freshly prepared lysis buffer and incubated for 10 min at 65 ºC, followed by addition of 3.5 μL of stop buffer.

    Lysis:

    Article Title: Effects of microvirin monomers and oligomers on hepatitis C virus
    Article Snippet: After 3 h of continuous shaking at 25°C, cells were harvested by centrifugation at 5000 g for 15 min, resuspended in a lysis buffer (50 mM Tris, 100 mM NaCl, 1% Triton X-100, pH 8.0) and disintegrated by sonication (40 on/off cycles with 20 μm amplitude for 15 s at 4°C). .. The resulting cell lysates were centrifuged at 20000 g for 30 min and the supernatants were loaded onto Ni2+ -nitrilotriacetate affinity chromatography columns (Ni-NTA) (Qiagen, 2 ml bed volume) equilibrated with washing buffer (50 mM Tris, 50 mM NaCl, pH 8.0).

    Article Title: Catalytic-Site Mutations in the MYST Family Histone Acetyltransferase Esa1
    Article Snippet: .. After centrifugation and flash freezing of cell pellets in a dry ice/ethanol bath, cell lysates were prepared under native conditions and incubated with Ni-NTA beads (QIAGEN, Valencia, CA; cat. no. 1018244) essentially according to the manufacturer's recommended protocol with the following modifications: lysis buffer was 50 m m NaH2 PO4 , 500 m m NaCl, 10 m m imidazole, 10% glycerol, 1% Triton X-100, 10 m m β-mercaptoethanol, pH 8.0; His6 -Esa1 was eluted in 100 μl fractions, and the peak His6 -Esa1 fraction (determined by SDS–PAGE and Coomassie staining) was used as described below; and all steps were performed at room temperature due to aggregation of Esa1 at 4°. .. His6 -Esa1 (2 μg) was then incubated with chicken core histones and [3 H]-acetyl CoA for 30 min at room temperature followed by separation by SDS–PAGE and fluorography to determine incorporation of [3 H]-acetyl groups into histones.

    Article Title: Epitope Mapping of Function-blocking Monoclonal Antibody CM6 Suggests a "Weak" Integrin Binding Site on the Laminin-332 LG2 Domain
    Article Snippet: Cell lysis was carried out by passage of cell suspension through French Press (SLM Instruments, Urbana, IL) at 1,500 psi twice. .. Clarified cell lysates were applied to Ni-NTA columns (Qiagen, Valencia, CA).

    Article Title: Preimplantation Genetic Testing for Monogenic Disease of Spinal Muscular Atrophy by Multiple Displacement Amplification: 11 unaffected livebirths
    Article Snippet: .. 2.6 Cell lysates and MDA protocol Cell lysis and MDA were performed using the reagents and protocol provided by the manufacturer of the Repli-g Midi kit (Qiagen, Germany). .. Samples were mixed with an additional 3.5 μL of freshly prepared lysis buffer and incubated for 10 min at 65 ºC, followed by addition of 3.5 μL of stop buffer.

    Article Title: Roles of the Linker Region of RNA Helicase A in HIV-1 RNA Metabolism
    Article Snippet: Briefly, cells were transfected with indicated plasmids using 25 kDa linear Polyethylenimine (PEI, pH7.0, Polysciences Inc.), collected 48 hours later, washed with ice-cold phosphate-buffered saline, and lysed in lysis buffer [50 mM NaH2 PO4 , pH 7.4, 300 mM NaCl, 10 mM imidazole, 0.5% Triton-X 100, 10% glycerol, and protease inhibitor cocktail tablets (Roche)]. .. The cell lysates were cleared by centrifugation and then incubated with Ni-nitrilotriacetic acid (NTA) agarose (Qiagen) at 4°C for 2 hours to capture His-tagged proteins.

    Article Title: SerpinA3N is a novel hypothalamic gene upregulated by a high-fat diet and leptin in mice
    Article Snippet: Real-time PCR After the challenge, the medium was removed and cells were lysed using RLT lysis buffer (RNeasy Mini Kit Qiagen, Venlo, The Netherlands). .. Cell lysates were then collected and RNA extracted using an RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions.

    Recombinant:

    Article Title: Effects of microvirin monomers and oligomers on hepatitis C virus
    Article Snippet: Paragraph title: Expression and purification of the recombinant MVN oligomers ... The resulting cell lysates were centrifuged at 20000 g for 30 min and the supernatants were loaded onto Ni2+ -nitrilotriacetate affinity chromatography columns (Ni-NTA) (Qiagen, 2 ml bed volume) equilibrated with washing buffer (50 mM Tris, 50 mM NaCl, pH 8.0).

    Article Title: Catalytic-Site Mutations in the MYST Family Histone Acetyltransferase Esa1
    Article Snippet: Paragraph title: Preparation of recombinant Esa1 and HAT assays: ... After centrifugation and flash freezing of cell pellets in a dry ice/ethanol bath, cell lysates were prepared under native conditions and incubated with Ni-NTA beads (QIAGEN, Valencia, CA; cat. no. 1018244) essentially according to the manufacturer's recommended protocol with the following modifications: lysis buffer was 50 m m NaH2 PO4 , 500 m m NaCl, 10 m m imidazole, 10% glycerol, 1% Triton X-100, 10 m m β-mercaptoethanol, pH 8.0; His6 -Esa1 was eluted in 100 μl fractions, and the peak His6 -Esa1 fraction (determined by SDS–PAGE and Coomassie staining) was used as described below; and all steps were performed at room temperature due to aggregation of Esa1 at 4°.

    Article Title: Roles of the Linker Region of RNA Helicase A in HIV-1 RNA Metabolism
    Article Snippet: The cell lysates were cleared by centrifugation and then incubated with Ni-nitrilotriacetic acid (NTA) agarose (Qiagen) at 4°C for 2 hours to capture His-tagged proteins. .. After extensive washing, recombinant proteins were eluted by 250 mM imidazole solution (pH 7.4).