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  • 94
    Millipore gel electrophoresis
    Gel Electrophoresis, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 862 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore gel electrophoresis sds page
    Reconstruction of the nucleoprotein complex on the LEE1 promoter. Protein crude extract was prepared from W3110 harboring pTB101 (-pch) or from pTB101- pch -FLAG (+pch) and was incubated with a DNA fragment of the LEE1 promoter immobilized on magnetic beads. A. Bound proteins were separated by <t>SDS-PAGE</t> and were visualized by silver staining, and major proteins were identified by LC-MS/MS. B. H-NS in the DNA-bound samples. H-NS in samples of the LEE1 promoter DNA (P LEE1 )-bound proteins (Bound) and crude protein extract (Input) were examined by immunoblotting using anti-H-NS antiserum. As a control, gadE promoter DNA (P gadE ) was used to isolate promoter bound proteins from the same extracts.
    Gel Electrophoresis Sds Page, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 263 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore sds polyacrylamide gel electrophoresis page gels
    Mapping of Hsp90–WT1 interacting domains by GST pull-down assay . (A) Schematic diagram of Hsp90 domains on the left, with the expression constructs and their binding to WT1 on the right. GST pull-down assays were performed using bacterially expressed GST-WT1 (1-517 aa) and GST (negative control) and in vitro–translated and 35 S-methionine–labeled full-length and deletion mutants of Hsp90. Bound proteins were separated by <t>SDS-PAGE</t> and visualized by fluorography. Bottom panel shows the in vitro–translated Hsp90 proteins. (B) Schematic diagram of WT1 domains on the left, with the expression constructs and their binding to Hsp90 on the right. GST pull-down assays were performed using bacterially expressed GST-Hsp90 (1-282 aa), GST (negative control), and in vitro–translated and 35 S-methionine–labeled full-length and deletion mutants of WT1 and analyzed in panel A. A vertical line has been inserted to indicate a repositioned gel lane in the bottom panel.
    Sds Polyacrylamide Gel Electrophoresis Page Gels, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1992 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad sds polyacrylamide gel electrophoresis page gels
    Characterization of bacterial strains. (a) Aliquots of strains M1, 12 and 57 GAS were incubated with normal human plasma, and bound proteins were eluted and analysed by <t>SDS–PAGE</t> and Western blotting using polyclonal anti-C4bp. The position of the 7 0 000 MW α-chain of C4bp is indicated by the arrow. Only strain M1 bound C4bp. (b) Aliquots of overnight culture supernatant from strains M1, M12 and M57 GAS were analysed by SDS-PAGE and Western blotting using polyclonal anti-M1 SIC. This showed that SIC is produced by strains M1 and M57 but not by M12. (c) Southern blot of Hin dIII digested streptococcal DNA from strains M1, 12 and 57 probed with labelled M1 sic PCR amplicon showing the presence of a sic or sic -like gene in all three GAS strains tested. No hybridizing bands were detected in either of the negative control Streptococci (data not shown).
    Sds Polyacrylamide Gel Electrophoresis Page Gels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 92 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher sds polyacrylamide gradient gel electrophoresis
    Characterization of bacterial strains. (a) Aliquots of strains M1, 12 and 57 GAS were incubated with normal human plasma, and bound proteins were eluted and analysed by <t>SDS–PAGE</t> and Western blotting using polyclonal anti-C4bp. The position of the 7 0 000 MW α-chain of C4bp is indicated by the arrow. Only strain M1 bound C4bp. (b) Aliquots of overnight culture supernatant from strains M1, M12 and M57 GAS were analysed by SDS-PAGE and Western blotting using polyclonal anti-M1 SIC. This showed that SIC is produced by strains M1 and M57 but not by M12. (c) Southern blot of Hin dIII digested streptococcal DNA from strains M1, 12 and 57 probed with labelled M1 sic PCR amplicon showing the presence of a sic or sic -like gene in all three GAS strains tested. No hybridizing bands were detected in either of the negative control Streptococci (data not shown).
    Sds Polyacrylamide Gradient Gel Electrophoresis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher sds polyacrylamide gel electrophoresis gel
    Characterization of bacterial strains. (a) Aliquots of strains M1, 12 and 57 GAS were incubated with normal human plasma, and bound proteins were eluted and analysed by <t>SDS–PAGE</t> and Western blotting using polyclonal anti-C4bp. The position of the 7 0 000 MW α-chain of C4bp is indicated by the arrow. Only strain M1 bound C4bp. (b) Aliquots of overnight culture supernatant from strains M1, M12 and M57 GAS were analysed by SDS-PAGE and Western blotting using polyclonal anti-M1 SIC. This showed that SIC is produced by strains M1 and M57 but not by M12. (c) Southern blot of Hin dIII digested streptococcal DNA from strains M1, 12 and 57 probed with labelled M1 sic PCR amplicon showing the presence of a sic or sic -like gene in all three GAS strains tested. No hybridizing bands were detected in either of the negative control Streptococci (data not shown).
    Sds Polyacrylamide Gel Electrophoresis Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 140 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jule Inc sds polyacrylamide gel electrophoresis gels
    Characterization of bacterial strains. (a) Aliquots of strains M1, 12 and 57 GAS were incubated with normal human plasma, and bound proteins were eluted and analysed by <t>SDS–PAGE</t> and Western blotting using polyclonal anti-C4bp. The position of the 7 0 000 MW α-chain of C4bp is indicated by the arrow. Only strain M1 bound C4bp. (b) Aliquots of overnight culture supernatant from strains M1, M12 and M57 GAS were analysed by SDS-PAGE and Western blotting using polyclonal anti-M1 SIC. This showed that SIC is produced by strains M1 and M57 but not by M12. (c) Southern blot of Hin dIII digested streptococcal DNA from strains M1, 12 and 57 probed with labelled M1 sic PCR amplicon showing the presence of a sic or sic -like gene in all three GAS strains tested. No hybridizing bands were detected in either of the negative control Streptococci (data not shown).
    Sds Polyacrylamide Gel Electrophoresis Gels, supplied by Jule Inc, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad sds polyacrylamid gel electrophoresis sds page
    Impact of NCT on purified Stx2 demonstrated in gel electrophoresis. (A) Purified Stx2 was treated with 55 mM NCT for 30 min at RT. Then, an aliquot was used for <t>SDS-PAGE.</t> Reducing buffer contained 4.5% v/v mercaptoethanol. A gel containing bands of aliquots containing 10 or 15 µg Stx2 is shown (one representative of three independent experiments). Bands a–f were subjected to in-gel digestion and mass spectrometry. (B) Purified Stx2 was treated with 55 mM NCT for 30 min at RT and subjected to a tricine gel for improved separation of the low molecular weight bands. Note the separation of the B subunit in bands a and b, which were subjected to in-gel digestion and mass spectrometry.
    Sds Polyacrylamid Gel Electrophoresis Sds Page, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    GE Healthcare sds polyacrylamide gel electrophoresis page gels
    Western blot analysis of expression of SF2/ASF in a clonal population of U2-OS cells stably expressing HPV-16 E2 protein. (A) Cell extracts in E buffer were prepared from U2-OS cells transformed with the E2 expression vector (E2) and from cells stably transformed with the vector alone (V). The protein concentration was measured by the Bradford assay, and equal quantities (20 μg) were electrophoresed in each <t>SDS-PAGE</t> track. Panels were Western blotted with monoclonal antibodies against the proteins shown to the right. Each experiment was carried out three times with very similar results. (B) Quantification by densitometry scanning of levels of SF2/ASF in pCMV-transformed (V) and pCMVE2-transformed (E2) U2-OS cells, showing the mean and standard deviation from the mean of three separate experiments.
    Sds Polyacrylamide Gel Electrophoresis Page Gels, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 89/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Reconstruction of the nucleoprotein complex on the LEE1 promoter. Protein crude extract was prepared from W3110 harboring pTB101 (-pch) or from pTB101- pch -FLAG (+pch) and was incubated with a DNA fragment of the LEE1 promoter immobilized on magnetic beads. A. Bound proteins were separated by SDS-PAGE and were visualized by silver staining, and major proteins were identified by LC-MS/MS. B. H-NS in the DNA-bound samples. H-NS in samples of the LEE1 promoter DNA (P LEE1 )-bound proteins (Bound) and crude protein extract (Input) were examined by immunoblotting using anti-H-NS antiserum. As a control, gadE promoter DNA (P gadE ) was used to isolate promoter bound proteins from the same extracts.

    Journal: PLoS ONE

    Article Title: Gene Activation through the Modulation of Nucleoid Structures by a Horizontally Transferred Regulator, Pch, in Enterohemorrhagic Escherichia coli

    doi: 10.1371/journal.pone.0149718

    Figure Lengend Snippet: Reconstruction of the nucleoprotein complex on the LEE1 promoter. Protein crude extract was prepared from W3110 harboring pTB101 (-pch) or from pTB101- pch -FLAG (+pch) and was incubated with a DNA fragment of the LEE1 promoter immobilized on magnetic beads. A. Bound proteins were separated by SDS-PAGE and were visualized by silver staining, and major proteins were identified by LC-MS/MS. B. H-NS in the DNA-bound samples. H-NS in samples of the LEE1 promoter DNA (P LEE1 )-bound proteins (Bound) and crude protein extract (Input) were examined by immunoblotting using anti-H-NS antiserum. As a control, gadE promoter DNA (P gadE ) was used to isolate promoter bound proteins from the same extracts.

    Article Snippet: The proteins were separated by SDS-polyacrylamide (12% or 10%) gel electrophoresis (SDS-PAGE) and were transferred onto an Immobilon membrane (Millipore).

    Techniques: Incubation, Magnetic Beads, SDS Page, Silver Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Mapping of Hsp90–WT1 interacting domains by GST pull-down assay . (A) Schematic diagram of Hsp90 domains on the left, with the expression constructs and their binding to WT1 on the right. GST pull-down assays were performed using bacterially expressed GST-WT1 (1-517 aa) and GST (negative control) and in vitro–translated and 35 S-methionine–labeled full-length and deletion mutants of Hsp90. Bound proteins were separated by SDS-PAGE and visualized by fluorography. Bottom panel shows the in vitro–translated Hsp90 proteins. (B) Schematic diagram of WT1 domains on the left, with the expression constructs and their binding to Hsp90 on the right. GST pull-down assays were performed using bacterially expressed GST-Hsp90 (1-282 aa), GST (negative control), and in vitro–translated and 35 S-methionine–labeled full-length and deletion mutants of WT1 and analyzed in panel A. A vertical line has been inserted to indicate a repositioned gel lane in the bottom panel.

    Journal: Blood

    Article Title: Heat shock protein 90 regulates the expression of Wilms tumor 1 protein in myeloid leukemias

    doi: 10.1182/blood-2009-10-247239

    Figure Lengend Snippet: Mapping of Hsp90–WT1 interacting domains by GST pull-down assay . (A) Schematic diagram of Hsp90 domains on the left, with the expression constructs and their binding to WT1 on the right. GST pull-down assays were performed using bacterially expressed GST-WT1 (1-517 aa) and GST (negative control) and in vitro–translated and 35 S-methionine–labeled full-length and deletion mutants of Hsp90. Bound proteins were separated by SDS-PAGE and visualized by fluorography. Bottom panel shows the in vitro–translated Hsp90 proteins. (B) Schematic diagram of WT1 domains on the left, with the expression constructs and their binding to Hsp90 on the right. GST pull-down assays were performed using bacterially expressed GST-Hsp90 (1-282 aa), GST (negative control), and in vitro–translated and 35 S-methionine–labeled full-length and deletion mutants of WT1 and analyzed in panel A. A vertical line has been inserted to indicate a repositioned gel lane in the bottom panel.

    Article Snippet: For Western blot analysis, cell extracts or immunoprecipitates were resolved on SDS–polyacrylamide gel electrophoresis (PAGE) gels and transferred to polyvinylidene difluoride membranes (Millipore).

    Techniques: Pull Down Assay, Expressing, Construct, Binding Assay, Negative Control, In Vitro, Labeling, SDS Page

    Pharmacologic inhibition of Hsp90 down-regulates WT1 protein . (A) K562 and (B) KG-1 leukemia cells were treated with the Hsp90 inhibitor 17-AAG for 24 hours. The cells were lysed, and protein extracts were subjected to SDS-PAGE and analyzed by Western blotting for WT1, Hsp90, and/or the WT1-regulated protein c-Myc. β-actin was used as loading control. (C) K562, (D) KG1, (E) Kasumi-1, and (F) MV4-11 leukemia cells were treated with the Hsp90 inhibitor STA-9090 for 24 hours and analyzed for WT1 expression by Western blotting. (G) Primary myeloid leukemia blasts from 5 AML patients (PS#1-5) were isolated, treated with STA-9090 for 24 hours, and analyzed for WT1 and β-actin by Western blotting.

    Journal: Blood

    Article Title: Heat shock protein 90 regulates the expression of Wilms tumor 1 protein in myeloid leukemias

    doi: 10.1182/blood-2009-10-247239

    Figure Lengend Snippet: Pharmacologic inhibition of Hsp90 down-regulates WT1 protein . (A) K562 and (B) KG-1 leukemia cells were treated with the Hsp90 inhibitor 17-AAG for 24 hours. The cells were lysed, and protein extracts were subjected to SDS-PAGE and analyzed by Western blotting for WT1, Hsp90, and/or the WT1-regulated protein c-Myc. β-actin was used as loading control. (C) K562, (D) KG1, (E) Kasumi-1, and (F) MV4-11 leukemia cells were treated with the Hsp90 inhibitor STA-9090 for 24 hours and analyzed for WT1 expression by Western blotting. (G) Primary myeloid leukemia blasts from 5 AML patients (PS#1-5) were isolated, treated with STA-9090 for 24 hours, and analyzed for WT1 and β-actin by Western blotting.

    Article Snippet: For Western blot analysis, cell extracts or immunoprecipitates were resolved on SDS–polyacrylamide gel electrophoresis (PAGE) gels and transferred to polyvinylidene difluoride membranes (Millipore).

    Techniques: Inhibition, SDS Page, Western Blot, Expressing, Isolation

    Direct interaction between WT1 and Hsp90 . (A) Equal amounts of K562 protein extracts were immunoprecipitated (IP) with agarose-conjugated mouse immunoglobulin G (IgG) or anti-Hsp90 antibodies, and immunoprecipitates were subjected to SDS-PAGE and immunoblotted (IB) for WT1 (top panel) and Hsp90 (bottom panel). Input represents ∼5% of the total protein extract used for immunoprecipitation. (B) Subcellular colocalization of WT1 and Hsp90. K562 cells were stained with DAPI (blue, nuclear stain) and antibodies to WT1 (green) or Hsp90 (red), and confocal images were acquired at 100× magnification. (C) GST pull-down assay. In vitro–translated and 35 S-methionine–labeled full-length Hsp90 was incubated with GST or GST-WT1 protein immobilized on glutathione-sepharose beads, and bound WT1 was detected by fluorography (top panel). 20% of the in vitro–translated protein was used for pull-downs. The bottom panel shows the purity of GST-fused proteins on a Coomassie blue-stained SDS-PAGE gel. (D) Dose-dependent binding of Hsp90 to WT1. Increasing amounts of 35 S-methionine–labeled Hsp90 were added to GST-WT1, and binding was analyzed by autoradiography.

    Journal: Blood

    Article Title: Heat shock protein 90 regulates the expression of Wilms tumor 1 protein in myeloid leukemias

    doi: 10.1182/blood-2009-10-247239

    Figure Lengend Snippet: Direct interaction between WT1 and Hsp90 . (A) Equal amounts of K562 protein extracts were immunoprecipitated (IP) with agarose-conjugated mouse immunoglobulin G (IgG) or anti-Hsp90 antibodies, and immunoprecipitates were subjected to SDS-PAGE and immunoblotted (IB) for WT1 (top panel) and Hsp90 (bottom panel). Input represents ∼5% of the total protein extract used for immunoprecipitation. (B) Subcellular colocalization of WT1 and Hsp90. K562 cells were stained with DAPI (blue, nuclear stain) and antibodies to WT1 (green) or Hsp90 (red), and confocal images were acquired at 100× magnification. (C) GST pull-down assay. In vitro–translated and 35 S-methionine–labeled full-length Hsp90 was incubated with GST or GST-WT1 protein immobilized on glutathione-sepharose beads, and bound WT1 was detected by fluorography (top panel). 20% of the in vitro–translated protein was used for pull-downs. The bottom panel shows the purity of GST-fused proteins on a Coomassie blue-stained SDS-PAGE gel. (D) Dose-dependent binding of Hsp90 to WT1. Increasing amounts of 35 S-methionine–labeled Hsp90 were added to GST-WT1, and binding was analyzed by autoradiography.

    Article Snippet: For Western blot analysis, cell extracts or immunoprecipitates were resolved on SDS–polyacrylamide gel electrophoresis (PAGE) gels and transferred to polyvinylidene difluoride membranes (Millipore).

    Techniques: Immunoprecipitation, SDS Page, Staining, Pull Down Assay, In Vitro, Labeling, Incubation, Binding Assay, Autoradiography

    Swo1p and Rng3p are required for the stability of myo2-E1p. (A) Lysates of the indicated strains were arrested at the restrictive temperature of 36°C for 5 h, resolved by SDS-PAGE (12% polyacrylamide), immunoblotted, and probed with antibodies

    Journal:

    Article Title: Hsp90 Protein in Fission Yeast Swo1p and UCS Protein Rng3p Facilitate Myosin II Assembly and Function

    doi: 10.1128/EC.4.3.567-576.2005

    Figure Lengend Snippet: Swo1p and Rng3p are required for the stability of myo2-E1p. (A) Lysates of the indicated strains were arrested at the restrictive temperature of 36°C for 5 h, resolved by SDS-PAGE (12% polyacrylamide), immunoblotted, and probed with antibodies

    Article Snippet: After the final wash, the beads were resuspended in SDS-polyacrylamide gel electrophoresis (PAGE) gel loading buffer and heated at 95°C for 5 min. Proteins were separated on SDS-8% polyacrylamide gels and transferred to a polyvinylidene difluoride sequencing. membrane (Millipore Corp., Bedford, Mass.).

    Techniques: SDS Page

    Upregulation of cellular RNR expression and activity by MCMV. (A) Cellular R1 and R2 levels during MCMV infection. NIH 3T3 cells were growth-arrested in 0.5% calf serum and then either infected with active or UV-irradiated MCMV (MOI, 5 PFU/cell) or mock-infected. Whole-cell extracts were prepared at various times after infection, separated by SDS-PAGE, and analyzed by immunoblotting with the anti-R1, anti-R2, and anti-actin antibodies. A sample from quiescent cells stimulated with 10% calf serum for 24 h was also included. Lanes: 1, mock infection; 2, 6 hpi; 3, 12 hpi; 4, 24 hpi; 5, 36 hpi; 6, 48 hpi; 7, 60 hpi; 8, UV-inactivated MCMV 48 hpi; 9, noninfected cells grown in the presence of 10% serum. (B) Effect of MCMV infection on dNTP pool sizes in resting cells. NIH 3T3 cells were growth arrested in 0.5% calf serum and then infected with active or UV-inactivated MCMV (MOI, 5 PFU/cell). After virus adsorption, cells were either treated with 0.5 mM HU or left untreated. The nucleotides were extracted at 48 hpi, and their levels were measured by high-performance liquid chromatography. Levels of nucleotide pools are expressed as percentages of the total nucleoside triphosphate pool (CTP + UTP + ATP + GTP + dCTP + dTTP + dATP + dGTP) to minimize variations due to small differences in cell number in the samples.

    Journal: Journal of Virology

    Article Title: The Ribonucleotide Reductase R1 Homolog of Murine Cytomegalovirus Is Not a Functional Enzyme Subunit but Is Required for Pathogenesis †

    doi: 10.1128/JVI.78.8.4278-4288.2004

    Figure Lengend Snippet: Upregulation of cellular RNR expression and activity by MCMV. (A) Cellular R1 and R2 levels during MCMV infection. NIH 3T3 cells were growth-arrested in 0.5% calf serum and then either infected with active or UV-irradiated MCMV (MOI, 5 PFU/cell) or mock-infected. Whole-cell extracts were prepared at various times after infection, separated by SDS-PAGE, and analyzed by immunoblotting with the anti-R1, anti-R2, and anti-actin antibodies. A sample from quiescent cells stimulated with 10% calf serum for 24 h was also included. Lanes: 1, mock infection; 2, 6 hpi; 3, 12 hpi; 4, 24 hpi; 5, 36 hpi; 6, 48 hpi; 7, 60 hpi; 8, UV-inactivated MCMV 48 hpi; 9, noninfected cells grown in the presence of 10% serum. (B) Effect of MCMV infection on dNTP pool sizes in resting cells. NIH 3T3 cells were growth arrested in 0.5% calf serum and then infected with active or UV-inactivated MCMV (MOI, 5 PFU/cell). After virus adsorption, cells were either treated with 0.5 mM HU or left untreated. The nucleotides were extracted at 48 hpi, and their levels were measured by high-performance liquid chromatography. Levels of nucleotide pools are expressed as percentages of the total nucleoside triphosphate pool (CTP + UTP + ATP + GTP + dCTP + dTTP + dATP + dGTP) to minimize variations due to small differences in cell number in the samples.

    Article Snippet: For immunoblotting, proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to Immobilon-P membranes (Millipore).

    Techniques: Expressing, Activity Assay, Infection, Irradiation, SDS Page, Adsorption, High Performance Liquid Chromatography

    (A) Time course of M45 expression during MCMV replication as determined by immunoblotting. Whole-cell lysates of mock-infected and MCMV-infected NIH 3T3 cells at various times after infection were separated by SDS-PAGE, transferred to a membrane, and probed with the anti-M45 antiserum or with the anti-actin monoclonal antibody. Lanes: 1, mock-infected cells; 2, 6 hpi; 3, 9 hpi; 4, 12 hpi; 5, 18 hpi; 6, 24 hpi; 7, 36 hpi; 8, 48 hpi. (B) Effect of PFA on M45 expression. Whole-cell extracts were prepared at 18 hpi (lanes 1 and 2), 24 hpi (lanes 3 and 4), or 48 hpi (lanes 5 and 6) from MCMV-infected NIH 3T3 cells treated with PFA (250 μg/ml) after virus adsorption (lanes 2, 4, and 6) or left untreated (lanes 1, 3, and 5). Protein expression was analyzed by immunoblotting with the anti-M45 antiserum or with the anti-actin monoclonal antibody. (C) Subcellular localization of M45 in MCMV-infected NIH 3T3 cells at 48 hpi, detected by immunofluorescence and confocal microscopy. Cells were incubated with the M45 antiserum and then with the secondary FITC-conjugated antibody. Nuclei were counterstained with propidium iodide. (D) Subcellular localization of transiently expressed M45 protein in NIH 3T3 cells transfected with the pcDNA3-45 vector at 24 h posttransfection. For immunofluorescence and confocal microscopy analysis, cells were incubated with the M45 antiserum and then with the secondary FITC-conjugated antibody. Nuclei were counterstained with propidium iodide. The merged pictures are shown.

    Journal: Journal of Virology

    Article Title: The Ribonucleotide Reductase R1 Homolog of Murine Cytomegalovirus Is Not a Functional Enzyme Subunit but Is Required for Pathogenesis †

    doi: 10.1128/JVI.78.8.4278-4288.2004

    Figure Lengend Snippet: (A) Time course of M45 expression during MCMV replication as determined by immunoblotting. Whole-cell lysates of mock-infected and MCMV-infected NIH 3T3 cells at various times after infection were separated by SDS-PAGE, transferred to a membrane, and probed with the anti-M45 antiserum or with the anti-actin monoclonal antibody. Lanes: 1, mock-infected cells; 2, 6 hpi; 3, 9 hpi; 4, 12 hpi; 5, 18 hpi; 6, 24 hpi; 7, 36 hpi; 8, 48 hpi. (B) Effect of PFA on M45 expression. Whole-cell extracts were prepared at 18 hpi (lanes 1 and 2), 24 hpi (lanes 3 and 4), or 48 hpi (lanes 5 and 6) from MCMV-infected NIH 3T3 cells treated with PFA (250 μg/ml) after virus adsorption (lanes 2, 4, and 6) or left untreated (lanes 1, 3, and 5). Protein expression was analyzed by immunoblotting with the anti-M45 antiserum or with the anti-actin monoclonal antibody. (C) Subcellular localization of M45 in MCMV-infected NIH 3T3 cells at 48 hpi, detected by immunofluorescence and confocal microscopy. Cells were incubated with the M45 antiserum and then with the secondary FITC-conjugated antibody. Nuclei were counterstained with propidium iodide. (D) Subcellular localization of transiently expressed M45 protein in NIH 3T3 cells transfected with the pcDNA3-45 vector at 24 h posttransfection. For immunofluorescence and confocal microscopy analysis, cells were incubated with the M45 antiserum and then with the secondary FITC-conjugated antibody. Nuclei were counterstained with propidium iodide. The merged pictures are shown.

    Article Snippet: For immunoblotting, proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to Immobilon-P membranes (Millipore).

    Techniques: Expressing, Infection, SDS Page, Adsorption, Immunofluorescence, Confocal Microscopy, Incubation, Transfection, Plasmid Preparation

    Identification of the MCMV M45 protein by the specific antiserum. (A) Immunoblot analysis of native or recombinant M45 expression in MCMV-infected NIH 3T3 cells, in cells transfected with an M45 expression vector, or in E. coli . Proteins of whole-cell lysates were separated by SDS-PAGE (5 to 15% acrylamide), transferred to a membrane, and probed with the M45 antiserum. Lanes: 1, extracts from mock-infected NIH 3T3 cells; 2, extracts from NIH 3T3 cells 48 h after infection with MCMV; 3, extracts from NIH 3T3 cells transiently transfected with the control vector pcDNA3; 4, extracts from NIH 3T3 cells transiently transfected with the M45 expression vector pcDNA3-M45; 5, extracts from IPTG-induced E. coli containing the M45 expression vector pET30-M45. (B) Immunoprecipitation of M45 by the specific antiserum. M45 was immunoprecipitated from cell extracts of MCMV-infected NIH 3T3 cells prepared at 48 hpi and was analyzed by immunoblotting with the M45 antiserum. Lanes: 1, cell extract from infected cells; 2, cell extract immunoprecipitated with the M45 antiserum; 3, cell extract immunoprecipitated with preimmune serum. Sizes of the molecular mass markers are shown on the left of each panel. Asterisk indicates the Ig heavy chains recognized by the secondary antibody.

    Journal: Journal of Virology

    Article Title: The Ribonucleotide Reductase R1 Homolog of Murine Cytomegalovirus Is Not a Functional Enzyme Subunit but Is Required for Pathogenesis †

    doi: 10.1128/JVI.78.8.4278-4288.2004

    Figure Lengend Snippet: Identification of the MCMV M45 protein by the specific antiserum. (A) Immunoblot analysis of native or recombinant M45 expression in MCMV-infected NIH 3T3 cells, in cells transfected with an M45 expression vector, or in E. coli . Proteins of whole-cell lysates were separated by SDS-PAGE (5 to 15% acrylamide), transferred to a membrane, and probed with the M45 antiserum. Lanes: 1, extracts from mock-infected NIH 3T3 cells; 2, extracts from NIH 3T3 cells 48 h after infection with MCMV; 3, extracts from NIH 3T3 cells transiently transfected with the control vector pcDNA3; 4, extracts from NIH 3T3 cells transiently transfected with the M45 expression vector pcDNA3-M45; 5, extracts from IPTG-induced E. coli containing the M45 expression vector pET30-M45. (B) Immunoprecipitation of M45 by the specific antiserum. M45 was immunoprecipitated from cell extracts of MCMV-infected NIH 3T3 cells prepared at 48 hpi and was analyzed by immunoblotting with the M45 antiserum. Lanes: 1, cell extract from infected cells; 2, cell extract immunoprecipitated with the M45 antiserum; 3, cell extract immunoprecipitated with preimmune serum. Sizes of the molecular mass markers are shown on the left of each panel. Asterisk indicates the Ig heavy chains recognized by the secondary antibody.

    Article Snippet: For immunoblotting, proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to Immobilon-P membranes (Millipore).

    Techniques: Recombinant, Expressing, Infection, Transfection, Plasmid Preparation, SDS Page, Immunoprecipitation

    Detection of M45 in purified MCMV particles by immunoblotting. Proteins from a whole-cell lysate of MCMV-infected NIH 3T3 cells at 48 hpi (lane 1) or from virus particles purified by two rounds of centrifugation through a 15% sucrose cushion (lane 2) or via density gradient centrifugation (lane 3) were separated by SDS-PAGE, blotted onto a membrane, and probed with antibodies against the viral proteins M45 and M44 and the cellular proteins R2 and actin.

    Journal: Journal of Virology

    Article Title: The Ribonucleotide Reductase R1 Homolog of Murine Cytomegalovirus Is Not a Functional Enzyme Subunit but Is Required for Pathogenesis †

    doi: 10.1128/JVI.78.8.4278-4288.2004

    Figure Lengend Snippet: Detection of M45 in purified MCMV particles by immunoblotting. Proteins from a whole-cell lysate of MCMV-infected NIH 3T3 cells at 48 hpi (lane 1) or from virus particles purified by two rounds of centrifugation through a 15% sucrose cushion (lane 2) or via density gradient centrifugation (lane 3) were separated by SDS-PAGE, blotted onto a membrane, and probed with antibodies against the viral proteins M45 and M44 and the cellular proteins R2 and actin.

    Article Snippet: For immunoblotting, proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to Immobilon-P membranes (Millipore).

    Techniques: Purification, Infection, Centrifugation, Gradient Centrifugation, SDS Page

    RNR assays. (A) SDS-PAGE analyses of purified recombinant M45. Lane 1, molecular mass markers at 203, 120, 90, and 51 kDa; lane 2, recombinant His-tagged M45 purified by chromatography on a nickel-agarose column followed by chromatography on a Superdex 200 column. (B) Catalytic activity of recombinant M45 assayed in the presence of an excess of mouse R2 protein (10 μg) before and after the addition of a constant amount of mouse R1 protein (2 μg). The increasing amounts of purified recombinant M45 are 0, 7, 14, and 28 μg. (C) Catalytic activity of recombinant M45 assayed in the presence of an excess of mouse R2 (10 μg) together with a constant amount of mouse R1 (2 μg) or of the catalytically inactive R1 C429A protein (9 μg). The increasing amounts of purified recombinant M45 are 0, 7, and 14 μg. (D) Allosteric inhibition of ATP-stimulated CDP reduction by dATP. Catalytic activity of the mouse complex R1-R2 alone or together with recombinant M45 protein was assayed in the presence of 0, 20, 80, or 400 μM dATP.

    Journal: Journal of Virology

    Article Title: The Ribonucleotide Reductase R1 Homolog of Murine Cytomegalovirus Is Not a Functional Enzyme Subunit but Is Required for Pathogenesis †

    doi: 10.1128/JVI.78.8.4278-4288.2004

    Figure Lengend Snippet: RNR assays. (A) SDS-PAGE analyses of purified recombinant M45. Lane 1, molecular mass markers at 203, 120, 90, and 51 kDa; lane 2, recombinant His-tagged M45 purified by chromatography on a nickel-agarose column followed by chromatography on a Superdex 200 column. (B) Catalytic activity of recombinant M45 assayed in the presence of an excess of mouse R2 protein (10 μg) before and after the addition of a constant amount of mouse R1 protein (2 μg). The increasing amounts of purified recombinant M45 are 0, 7, 14, and 28 μg. (C) Catalytic activity of recombinant M45 assayed in the presence of an excess of mouse R2 (10 μg) together with a constant amount of mouse R1 (2 μg) or of the catalytically inactive R1 C429A protein (9 μg). The increasing amounts of purified recombinant M45 are 0, 7, and 14 μg. (D) Allosteric inhibition of ATP-stimulated CDP reduction by dATP. Catalytic activity of the mouse complex R1-R2 alone or together with recombinant M45 protein was assayed in the presence of 0, 20, 80, or 400 μM dATP.

    Article Snippet: For immunoblotting, proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to Immobilon-P membranes (Millipore).

    Techniques: SDS Page, Purification, Recombinant, Chromatography, Activity Assay, Inhibition

    Certain PRG/IRGs are significantly expressed in the presence of FVP during a mitogenic response. (A) Effects of FVP in the cell cycle were determined by FACS of Propidium Iodide(PI)stained cells. The percent of cells in S phase is indicated at the top of each panel. BJ-TERT fibroblasts were treated with either DMSO or 300 nM FVP followed by serum stimulation for 0, 14, 22 and 30 h and stained with PI. (B) Western blot analysis of cell cycle progression, transcription markers and MCL1. BJ-TERT fibroblasts were harvested and lysed. Ten microgram of protein was resolved by 8% polyacrylamide/SDS gel electrophoresis, transferred to a PVDF membrane and specific antibodies were used to detect proteins, as indicated above. (C) 300 nM FVP inhibits phosphorylation of the CTD of RNAPII on Ser-2 and Ser-5 without affecting the phosphorylation state of pocket proteins. Exponentially growing BJ-TERT fibroblasts were treated with FVP at 10 nM, 30 nM, 100 nM, 300 nM, 1 μM and 10 μM for 2 and 4 h. Whole cell lysates were resolved by 6% SDS/PAGE and immunoblotted with antibodies to the indicated proteins/phosphorylation sites. Solid and dashed arrows at the bottom indicate the concentration of FVP leading to dephosphorylation of RNAPII and pocket proteins, respectively. The asterisk indicates a crossreacting band recognized by the anti-p130 antibody. (D) mRNA levels of selected genes at the indicated time points were detected by Q-RT-PCR. The results of three different experiments are shown. Data are represented as a fold change value of levels normalized to 1 at time zero. The results of three different experiments are shown.

    Journal: Cell Division

    Article Title: Complex effects of flavopiridol on the expression of primary response genes

    doi: 10.1186/1747-1028-7-11

    Figure Lengend Snippet: Certain PRG/IRGs are significantly expressed in the presence of FVP during a mitogenic response. (A) Effects of FVP in the cell cycle were determined by FACS of Propidium Iodide(PI)stained cells. The percent of cells in S phase is indicated at the top of each panel. BJ-TERT fibroblasts were treated with either DMSO or 300 nM FVP followed by serum stimulation for 0, 14, 22 and 30 h and stained with PI. (B) Western blot analysis of cell cycle progression, transcription markers and MCL1. BJ-TERT fibroblasts were harvested and lysed. Ten microgram of protein was resolved by 8% polyacrylamide/SDS gel electrophoresis, transferred to a PVDF membrane and specific antibodies were used to detect proteins, as indicated above. (C) 300 nM FVP inhibits phosphorylation of the CTD of RNAPII on Ser-2 and Ser-5 without affecting the phosphorylation state of pocket proteins. Exponentially growing BJ-TERT fibroblasts were treated with FVP at 10 nM, 30 nM, 100 nM, 300 nM, 1 μM and 10 μM for 2 and 4 h. Whole cell lysates were resolved by 6% SDS/PAGE and immunoblotted with antibodies to the indicated proteins/phosphorylation sites. Solid and dashed arrows at the bottom indicate the concentration of FVP leading to dephosphorylation of RNAPII and pocket proteins, respectively. The asterisk indicates a crossreacting band recognized by the anti-p130 antibody. (D) mRNA levels of selected genes at the indicated time points were detected by Q-RT-PCR. The results of three different experiments are shown. Data are represented as a fold change value of levels normalized to 1 at time zero. The results of three different experiments are shown.

    Article Snippet: Proteins were resolved by 8% polyacrylamide/SDS gel electrophoresis, and transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon-FL, Millipore) in 10 mM CAPS/10% methanol buffer (pH 11).

    Techniques: FACS, Staining, Western Blot, SDS-Gel, Electrophoresis, SDS Page, Concentration Assay, De-Phosphorylation Assay, Reverse Transcription Polymerase Chain Reaction

    Characterization of bacterial strains. (a) Aliquots of strains M1, 12 and 57 GAS were incubated with normal human plasma, and bound proteins were eluted and analysed by SDS–PAGE and Western blotting using polyclonal anti-C4bp. The position of the 7 0 000 MW α-chain of C4bp is indicated by the arrow. Only strain M1 bound C4bp. (b) Aliquots of overnight culture supernatant from strains M1, M12 and M57 GAS were analysed by SDS-PAGE and Western blotting using polyclonal anti-M1 SIC. This showed that SIC is produced by strains M1 and M57 but not by M12. (c) Southern blot of Hin dIII digested streptococcal DNA from strains M1, 12 and 57 probed with labelled M1 sic PCR amplicon showing the presence of a sic or sic -like gene in all three GAS strains tested. No hybridizing bands were detected in either of the negative control Streptococci (data not shown).

    Journal: Immunology

    Article Title: Streptococcal inhibitor of complement (SIC) inhibits the membrane attack complex by preventing uptake of C567 onto cell membranes

    doi: 10.1046/j.1365-2567.2001.01249.x

    Figure Lengend Snippet: Characterization of bacterial strains. (a) Aliquots of strains M1, 12 and 57 GAS were incubated with normal human plasma, and bound proteins were eluted and analysed by SDS–PAGE and Western blotting using polyclonal anti-C4bp. The position of the 7 0 000 MW α-chain of C4bp is indicated by the arrow. Only strain M1 bound C4bp. (b) Aliquots of overnight culture supernatant from strains M1, M12 and M57 GAS were analysed by SDS-PAGE and Western blotting using polyclonal anti-M1 SIC. This showed that SIC is produced by strains M1 and M57 but not by M12. (c) Southern blot of Hin dIII digested streptococcal DNA from strains M1, 12 and 57 probed with labelled M1 sic PCR amplicon showing the presence of a sic or sic -like gene in all three GAS strains tested. No hybridizing bands were detected in either of the negative control Streptococci (data not shown).

    Article Snippet: Samples were run on 15%, 10% or 6% SDS–polyacrylamide gel electrophoresis (PAGE) gels for C3, factor H or C4bp, respectively, in a Mini-PROTEAN II system (Bio-Rad Laboratories Ltd, Hemel Hempstead, UK), then electroblotted onto nitrocellulose.

    Techniques: Incubation, SDS Page, Western Blot, Produced, Southern Blot, Polymerase Chain Reaction, Amplification, Negative Control

    Impact of NCT on purified Stx2 demonstrated in gel electrophoresis. (A) Purified Stx2 was treated with 55 mM NCT for 30 min at RT. Then, an aliquot was used for SDS-PAGE. Reducing buffer contained 4.5% v/v mercaptoethanol. A gel containing bands of aliquots containing 10 or 15 µg Stx2 is shown (one representative of three independent experiments). Bands a–f were subjected to in-gel digestion and mass spectrometry. (B) Purified Stx2 was treated with 55 mM NCT for 30 min at RT and subjected to a tricine gel for improved separation of the low molecular weight bands. Note the separation of the B subunit in bands a and b, which were subjected to in-gel digestion and mass spectrometry.

    Journal: PLoS ONE

    Article Title: N-Chlorotaurine, a Long-Lived Oxidant Produced by Human Leukocytes, Inactivates Shiga Toxin of Enterohemorrhagic Escherichia coli

    doi: 10.1371/journal.pone.0047105

    Figure Lengend Snippet: Impact of NCT on purified Stx2 demonstrated in gel electrophoresis. (A) Purified Stx2 was treated with 55 mM NCT for 30 min at RT. Then, an aliquot was used for SDS-PAGE. Reducing buffer contained 4.5% v/v mercaptoethanol. A gel containing bands of aliquots containing 10 or 15 µg Stx2 is shown (one representative of three independent experiments). Bands a–f were subjected to in-gel digestion and mass spectrometry. (B) Purified Stx2 was treated with 55 mM NCT for 30 min at RT and subjected to a tricine gel for improved separation of the low molecular weight bands. Note the separation of the B subunit in bands a and b, which were subjected to in-gel digestion and mass spectrometry.

    Article Snippet: Structural Changes of Stx2 by NCT Evaluated by SDS-polyacrylamid Gel Electrophoresis (SDS-PAGE) An electrophoresis system from Biorad was used to perform SDS-PAGE with a 16% gel.

    Techniques: Purification, Nucleic Acid Electrophoresis, SDS Page, Mass Spectrometry, Molecular Weight

    Western blot analysis of expression of SF2/ASF in a clonal population of U2-OS cells stably expressing HPV-16 E2 protein. (A) Cell extracts in E buffer were prepared from U2-OS cells transformed with the E2 expression vector (E2) and from cells stably transformed with the vector alone (V). The protein concentration was measured by the Bradford assay, and equal quantities (20 μg) were electrophoresed in each SDS-PAGE track. Panels were Western blotted with monoclonal antibodies against the proteins shown to the right. Each experiment was carried out three times with very similar results. (B) Quantification by densitometry scanning of levels of SF2/ASF in pCMV-transformed (V) and pCMVE2-transformed (E2) U2-OS cells, showing the mean and standard deviation from the mean of three separate experiments.

    Journal: Journal of Virology

    Article Title: SF2/ASF Binds the Human Papillomavirus Type 16 Late RNA Control Element and Is Regulated during Differentiation of Virus-Infected Epithelial Cells

    doi: 10.1128/JVI.78.19.10598-10605.2004

    Figure Lengend Snippet: Western blot analysis of expression of SF2/ASF in a clonal population of U2-OS cells stably expressing HPV-16 E2 protein. (A) Cell extracts in E buffer were prepared from U2-OS cells transformed with the E2 expression vector (E2) and from cells stably transformed with the vector alone (V). The protein concentration was measured by the Bradford assay, and equal quantities (20 μg) were electrophoresed in each SDS-PAGE track. Panels were Western blotted with monoclonal antibodies against the proteins shown to the right. Each experiment was carried out three times with very similar results. (B) Quantification by densitometry scanning of levels of SF2/ASF in pCMV-transformed (V) and pCMVE2-transformed (E2) U2-OS cells, showing the mean and standard deviation from the mean of three separate experiments.

    Article Snippet: For Western blot analysis, proteins fractionated on SDS-polyacrylamide gel electrophoresis (PAGE) gels were electroblotted onto polyvinylidene difluoride membranes (Amersham).

    Techniques: Western Blot, Expressing, Stable Transfection, Transformation Assay, Plasmid Preparation, Protein Concentration, Bradford Assay, SDS Page, Standard Deviation