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  • 95
    Millipore sds polyacrylamide gel electrophoresis
    TCFβ1 is phosphorylated in vitro in an activation-dependent manner. Full-length TCFβ1 (residues 1 to 301) expressed as a GST fusion protein was incubated with cell extracts from Jurkat cells activated with PMA (5 ng/ml), PMA (5 ng/ml) plus PHA (5 μg/ml), or UV for the indicated lengths of time, processed for in vitro kinase assay, analyzed by <t>SDS-PAGE,</t> and visualized by autoradiography. GST alone was used as a negative control.
    Sds Polyacrylamide Gel Electrophoresis, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 10352 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher sds buffer
    Bak undergoes tyrosine dephosphorylated during the initiation of apoptosis. ( A ) 2D gel analysis of Bak from HT1080 cells using narrow-range linear pI gradient IPG-focusing strips and 15% <t>Tris-glycine</t> <t>SDS–PAGE</t> showed partial dephosphorylation after UV treatment, complete dephosphorylation was achieved by λ-phosphatase treatment. ( B ) 2D gel analysis of Bak of mitochondrial extracts from HT1080 cells treated either with ser/thr (PP1) or tyr (YOP) phosphatase, which resulted in new Bak species (arrowed). ( C ) Histograms of FACS analysis of Bak Ab-1-specific fluorescence in control untreated HT1080 cells, HT1080 cells±pre-treatment for 30 min before UV treatment with phophatase inhibitors sodium stibogluconate (SS; 110 μM), phenylarsine oxide (PAO; 5 μM), Cyclosporin A (75 μM) and Calyculin A (2 nM). Samples were analysed 4 h after UV damage. ( D ) Quantification of the increase in Bak Ab-1-specific fluorescence±4 h apoptotic stimuli camptothecin (CPT; 6 μM), Etoposide (EP; 10 μM), Staurosporine (STS; 100 nM) or 5 mJ/cm 2 UV in the presence and absence of tyrosine phoshatase inhibitors 110 μM SS or 5 μM PAO with the y -axis showing levels of Bak-specific fluorescence and the x -axis showing treatment conditions.
    Sds Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 779 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cell Signaling Technology Inc sds gel electrophoresis
    Bak undergoes tyrosine dephosphorylated during the initiation of apoptosis. ( A ) 2D gel analysis of Bak from HT1080 cells using narrow-range linear pI gradient IPG-focusing strips and 15% <t>Tris-glycine</t> <t>SDS–PAGE</t> showed partial dephosphorylation after UV treatment, complete dephosphorylation was achieved by λ-phosphatase treatment. ( B ) 2D gel analysis of Bak of mitochondrial extracts from HT1080 cells treated either with ser/thr (PP1) or tyr (YOP) phosphatase, which resulted in new Bak species (arrowed). ( C ) Histograms of FACS analysis of Bak Ab-1-specific fluorescence in control untreated HT1080 cells, HT1080 cells±pre-treatment for 30 min before UV treatment with phophatase inhibitors sodium stibogluconate (SS; 110 μM), phenylarsine oxide (PAO; 5 μM), Cyclosporin A (75 μM) and Calyculin A (2 nM). Samples were analysed 4 h after UV damage. ( D ) Quantification of the increase in Bak Ab-1-specific fluorescence±4 h apoptotic stimuli camptothecin (CPT; 6 μM), Etoposide (EP; 10 μM), Staurosporine (STS; 100 nM) or 5 mJ/cm 2 UV in the presence and absence of tyrosine phoshatase inhibitors 110 μM SS or 5 μM PAO with the y -axis showing levels of Bak-specific fluorescence and the x -axis showing treatment conditions.
    Sds Gel Electrophoresis, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    GE Healthcare sds page gel electrophoresis gels
    Phosphor imager picture of <t>SDS-PAGE</t> gel of the product from the 125 I-cetuximab labelling reaction. The 125 I-cetuximab labelling reaction with 73.1, 112.2 and 150.0 MBq with miniaturized IODOGEN-coated mAb method demonstrating unaffected integrity with respect to the molecular weight. Conditions: 50 μg (0.33 nmol) cetuximab, 2.5 μg (5.7 nmol) IODOGEN, 250 μL reaction volume, 90-s reaction time.
    Sds Page Gel Electrophoresis Gels, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 83/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher sds page gel electrophoresis
    MALDI-TOF spectrum of Bovine SP-B dimer sample eluted from <t>SDS</t> gel of ETS-exposed surfactant sample. The ABI Voyager-DE STR MALDI-TOF was run in linear mode with delayed extraction and negative polarity. The instrument had an accelerating voltage of 2000
    Sds Page Gel Electrophoresis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 456 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Thermo Fisher sds nupage gel electrophoresis
    MALDI-TOF spectrum of Bovine SP-B dimer sample eluted from <t>SDS</t> gel of ETS-exposed surfactant sample. The ABI Voyager-DE STR MALDI-TOF was run in linear mode with delayed extraction and negative polarity. The instrument had an accelerating voltage of 2000
    Sds Nupage Gel Electrophoresis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 79/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Beyotime sds page gel electrophoresis
    MALDI-TOF spectrum of Bovine SP-B dimer sample eluted from <t>SDS</t> gel of ETS-exposed surfactant sample. The ABI Voyager-DE STR MALDI-TOF was run in linear mode with delayed extraction and negative polarity. The instrument had an accelerating voltage of 2000
    Sds Page Gel Electrophoresis, supplied by Beyotime, used in various techniques. Bioz Stars score: 88/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Servicebio Inc sds page gel electrophoresis
    MALDI-TOF spectrum of Bovine SP-B dimer sample eluted from <t>SDS</t> gel of ETS-exposed surfactant sample. The ABI Voyager-DE STR MALDI-TOF was run in linear mode with delayed extraction and negative polarity. The instrument had an accelerating voltage of 2000
    Sds Page Gel Electrophoresis, supplied by Servicebio Inc, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    83
    Thermo Fisher sds gel electrophoreses
    MALDI-TOF spectrum of Bovine SP-B dimer sample eluted from <t>SDS</t> gel of ETS-exposed surfactant sample. The ABI Voyager-DE STR MALDI-TOF was run in linear mode with delayed extraction and negative polarity. The instrument had an accelerating voltage of 2000
    Sds Gel Electrophoreses, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 83/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Thermo Fisher sds poly acrylamide gel electrophoresis
    <t>SDS-PAGE</t> and Western blot analysis of a serum sample (left) and milk sample (right), as indicated. The serum sample was diluted 1:1600 and the milk sample was diluted 1:40. In both cases, the purified standard mink IgG preparation was also included. All samples were electrophoresed under reducing conditions on 12% NuPAGE Bis–Tris gel as described in “ Methods ” section. For Western blotting (WB), the blot was developed with polyclonal goat anti-ferret IgG, followed by alkaline phosphatase-conjugated rabbit anti-goat antibody (see “ Methods ” section). SDS-PAGE: Lane 1: molecular weight marker; Lane 2: serum/milk sample; Lane 3: purified standard mink IgG. WB: Lane 4: molecular weight marker; Lane 5: serum/milk sample; Lane 6: purified standard mink IgG. The positions of the molecular weight marker proteins (20–100 kDa) are indicated to the left of the gels and blots
    Sds Poly Acrylamide Gel Electrophoresis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 80/100, based on 152 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Bio-Rad sds poly acrylamide gel electrophoresis
    IRF-3 5D-inducible expression of ISG56 gene. rtTA-Neo (lanes 1 to 5) and rtTA-IRF-3 5D (lanes 6 to 10) Jurkat cells were exposed to DOX (1 μg/ml) and anti-IFN antibodies for the indicated times. Whole-cell extracts (50 μg) were subjected to <t>SDS-PAGE</t> and analyzed by immunoblotting with anti-ISG56 antibodies. Membranes were stripped and reprobed with anti-IRF-3 and anti-actin antibodies.
    Sds Poly Acrylamide Gel Electrophoresis, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 84/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    82
    Bio-Rad stain free sds gel electrophoresis gels
    IRF-3 5D-inducible expression of ISG56 gene. rtTA-Neo (lanes 1 to 5) and rtTA-IRF-3 5D (lanes 6 to 10) Jurkat cells were exposed to DOX (1 μg/ml) and anti-IFN antibodies for the indicated times. Whole-cell extracts (50 μg) were subjected to <t>SDS-PAGE</t> and analyzed by immunoblotting with anti-ISG56 antibodies. Membranes were stripped and reprobed with anti-IRF-3 and anti-actin antibodies.
    Stain Free Sds Gel Electrophoresis Gels, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 82/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    GE Healthcare one dimensional sds gel electrophoresis
    ARF-GAPs mediate Arf1 Δ N17p-Q71L and coatomer binding to the cytosolic domains of v-SNAREs. <t>SDS-PAGE</t> analysis of in vitro binding of coatomer (top) and Arf1ΔN17p-Q71L (bottom) to GST (lanes 1 and 2) or GST fusions to v-SNAREs (lanes 3 and 4, Bet1p-GST; lanes 5 and 6, Sec22p-GST; lanes 7 and 8, GST-Bos1p). GST or GST fusion proteins were immobilized onto glutathione-agarose. Where indicated, 20 nM Glo3p (A) or Gcs1p (B) were added to the reaction, and 50% of the amount added is shown in lane 9. The guanine nucleotide on Arf1ΔN17p-Q71L was exchanged to GTP before the binding reaction, and 1.2 μM preexchanged Arf1ΔN17p-Q71L was added to the binding reaction. All reactions contained GTP. The coatomer concentration was 40 nM in the assay. After the binding reaction, the unbound proteins were removed by centrifugation. The proteins immobilized on the glutathione-agarose were separated by SDS-PAGE followed by <t>SyproRed</t> staining and analysis using the red fluorescent mode of a Storm PhosphorImager. In A, lanes 10 and 11 represent 20% of Arf1ΔN17p-Q71L and 50% of coatomer present in the reaction, respectively. In Bet1p-GST and Sec22p-GST, GST is the COOH-terminal fusion partner, whereas in GST-Bos1p it is in the NH 2 -terminal position.
    One Dimensional Sds Gel Electrophoresis, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad sds gel electrophoresis
    ARF-GAPs mediate Arf1 Δ N17p-Q71L and coatomer binding to the cytosolic domains of v-SNAREs. <t>SDS-PAGE</t> analysis of in vitro binding of coatomer (top) and Arf1ΔN17p-Q71L (bottom) to GST (lanes 1 and 2) or GST fusions to v-SNAREs (lanes 3 and 4, Bet1p-GST; lanes 5 and 6, Sec22p-GST; lanes 7 and 8, GST-Bos1p). GST or GST fusion proteins were immobilized onto glutathione-agarose. Where indicated, 20 nM Glo3p (A) or Gcs1p (B) were added to the reaction, and 50% of the amount added is shown in lane 9. The guanine nucleotide on Arf1ΔN17p-Q71L was exchanged to GTP before the binding reaction, and 1.2 μM preexchanged Arf1ΔN17p-Q71L was added to the binding reaction. All reactions contained GTP. The coatomer concentration was 40 nM in the assay. After the binding reaction, the unbound proteins were removed by centrifugation. The proteins immobilized on the glutathione-agarose were separated by SDS-PAGE followed by <t>SyproRed</t> staining and analysis using the red fluorescent mode of a Storm PhosphorImager. In A, lanes 10 and 11 represent 20% of Arf1ΔN17p-Q71L and 50% of coatomer present in the reaction, respectively. In Bet1p-GST and Sec22p-GST, GST is the COOH-terminal fusion partner, whereas in GST-Bos1p it is in the NH 2 -terminal position.
    Sds Gel Electrophoresis, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 616 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Schuell GmbH sds gel electrophoresis
    ARF-GAPs mediate Arf1 Δ N17p-Q71L and coatomer binding to the cytosolic domains of v-SNAREs. <t>SDS-PAGE</t> analysis of in vitro binding of coatomer (top) and Arf1ΔN17p-Q71L (bottom) to GST (lanes 1 and 2) or GST fusions to v-SNAREs (lanes 3 and 4, Bet1p-GST; lanes 5 and 6, Sec22p-GST; lanes 7 and 8, GST-Bos1p). GST or GST fusion proteins were immobilized onto glutathione-agarose. Where indicated, 20 nM Glo3p (A) or Gcs1p (B) were added to the reaction, and 50% of the amount added is shown in lane 9. The guanine nucleotide on Arf1ΔN17p-Q71L was exchanged to GTP before the binding reaction, and 1.2 μM preexchanged Arf1ΔN17p-Q71L was added to the binding reaction. All reactions contained GTP. The coatomer concentration was 40 nM in the assay. After the binding reaction, the unbound proteins were removed by centrifugation. The proteins immobilized on the glutathione-agarose were separated by SDS-PAGE followed by <t>SyproRed</t> staining and analysis using the red fluorescent mode of a Storm PhosphorImager. In A, lanes 10 and 11 represent 20% of Arf1ΔN17p-Q71L and 50% of coatomer present in the reaction, respectively. In Bet1p-GST and Sec22p-GST, GST is the COOH-terminal fusion partner, whereas in GST-Bos1p it is in the NH 2 -terminal position.
    Sds Gel Electrophoresis, supplied by Schuell GmbH, used in various techniques. Bioz Stars score: 88/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA sds gel electrophoresis
    ARF-GAPs mediate Arf1 Δ N17p-Q71L and coatomer binding to the cytosolic domains of v-SNAREs. <t>SDS-PAGE</t> analysis of in vitro binding of coatomer (top) and Arf1ΔN17p-Q71L (bottom) to GST (lanes 1 and 2) or GST fusions to v-SNAREs (lanes 3 and 4, Bet1p-GST; lanes 5 and 6, Sec22p-GST; lanes 7 and 8, GST-Bos1p). GST or GST fusion proteins were immobilized onto glutathione-agarose. Where indicated, 20 nM Glo3p (A) or Gcs1p (B) were added to the reaction, and 50% of the amount added is shown in lane 9. The guanine nucleotide on Arf1ΔN17p-Q71L was exchanged to GTP before the binding reaction, and 1.2 μM preexchanged Arf1ΔN17p-Q71L was added to the binding reaction. All reactions contained GTP. The coatomer concentration was 40 nM in the assay. After the binding reaction, the unbound proteins were removed by centrifugation. The proteins immobilized on the glutathione-agarose were separated by SDS-PAGE followed by <t>SyproRed</t> staining and analysis using the red fluorescent mode of a Storm PhosphorImager. In A, lanes 10 and 11 represent 20% of Arf1ΔN17p-Q71L and 50% of coatomer present in the reaction, respectively. In Bet1p-GST and Sec22p-GST, GST is the COOH-terminal fusion partner, whereas in GST-Bos1p it is in the NH 2 -terminal position.
    Sds Gel Electrophoresis, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Biometra sds gel electrophoresis
    ARF-GAPs mediate Arf1 Δ N17p-Q71L and coatomer binding to the cytosolic domains of v-SNAREs. <t>SDS-PAGE</t> analysis of in vitro binding of coatomer (top) and Arf1ΔN17p-Q71L (bottom) to GST (lanes 1 and 2) or GST fusions to v-SNAREs (lanes 3 and 4, Bet1p-GST; lanes 5 and 6, Sec22p-GST; lanes 7 and 8, GST-Bos1p). GST or GST fusion proteins were immobilized onto glutathione-agarose. Where indicated, 20 nM Glo3p (A) or Gcs1p (B) were added to the reaction, and 50% of the amount added is shown in lane 9. The guanine nucleotide on Arf1ΔN17p-Q71L was exchanged to GTP before the binding reaction, and 1.2 μM preexchanged Arf1ΔN17p-Q71L was added to the binding reaction. All reactions contained GTP. The coatomer concentration was 40 nM in the assay. After the binding reaction, the unbound proteins were removed by centrifugation. The proteins immobilized on the glutathione-agarose were separated by SDS-PAGE followed by <t>SyproRed</t> staining and analysis using the red fluorescent mode of a Storm PhosphorImager. In A, lanes 10 and 11 represent 20% of Arf1ΔN17p-Q71L and 50% of coatomer present in the reaction, respectively. In Bet1p-GST and Sec22p-GST, GST is the COOH-terminal fusion partner, whereas in GST-Bos1p it is in the NH 2 -terminal position.
    Sds Gel Electrophoresis, supplied by Biometra, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Fisher Scientific sds gel electrophoresis
    ARF-GAPs mediate Arf1 Δ N17p-Q71L and coatomer binding to the cytosolic domains of v-SNAREs. <t>SDS-PAGE</t> analysis of in vitro binding of coatomer (top) and Arf1ΔN17p-Q71L (bottom) to GST (lanes 1 and 2) or GST fusions to v-SNAREs (lanes 3 and 4, Bet1p-GST; lanes 5 and 6, Sec22p-GST; lanes 7 and 8, GST-Bos1p). GST or GST fusion proteins were immobilized onto glutathione-agarose. Where indicated, 20 nM Glo3p (A) or Gcs1p (B) were added to the reaction, and 50% of the amount added is shown in lane 9. The guanine nucleotide on Arf1ΔN17p-Q71L was exchanged to GTP before the binding reaction, and 1.2 μM preexchanged Arf1ΔN17p-Q71L was added to the binding reaction. All reactions contained GTP. The coatomer concentration was 40 nM in the assay. After the binding reaction, the unbound proteins were removed by centrifugation. The proteins immobilized on the glutathione-agarose were separated by SDS-PAGE followed by <t>SyproRed</t> staining and analysis using the red fluorescent mode of a Storm PhosphorImager. In A, lanes 10 and 11 represent 20% of Arf1ΔN17p-Q71L and 50% of coatomer present in the reaction, respectively. In Bet1p-GST and Sec22p-GST, GST is the COOH-terminal fusion partner, whereas in GST-Bos1p it is in the NH 2 -terminal position.
    Sds Gel Electrophoresis, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 90/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Carl Roth GmbH sds gel electrophoresis
    ARF-GAPs mediate Arf1 Δ N17p-Q71L and coatomer binding to the cytosolic domains of v-SNAREs. <t>SDS-PAGE</t> analysis of in vitro binding of coatomer (top) and Arf1ΔN17p-Q71L (bottom) to GST (lanes 1 and 2) or GST fusions to v-SNAREs (lanes 3 and 4, Bet1p-GST; lanes 5 and 6, Sec22p-GST; lanes 7 and 8, GST-Bos1p). GST or GST fusion proteins were immobilized onto glutathione-agarose. Where indicated, 20 nM Glo3p (A) or Gcs1p (B) were added to the reaction, and 50% of the amount added is shown in lane 9. The guanine nucleotide on Arf1ΔN17p-Q71L was exchanged to GTP before the binding reaction, and 1.2 μM preexchanged Arf1ΔN17p-Q71L was added to the binding reaction. All reactions contained GTP. The coatomer concentration was 40 nM in the assay. After the binding reaction, the unbound proteins were removed by centrifugation. The proteins immobilized on the glutathione-agarose were separated by SDS-PAGE followed by <t>SyproRed</t> staining and analysis using the red fluorescent mode of a Storm PhosphorImager. In A, lanes 10 and 11 represent 20% of Arf1ΔN17p-Q71L and 50% of coatomer present in the reaction, respectively. In Bet1p-GST and Sec22p-GST, GST is the COOH-terminal fusion partner, whereas in GST-Bos1p it is in the NH 2 -terminal position.
    Sds Gel Electrophoresis, supplied by Carl Roth GmbH, used in various techniques. Bioz Stars score: 88/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Abcam sds gel electrophoresis
    ARF-GAPs mediate Arf1 Δ N17p-Q71L and coatomer binding to the cytosolic domains of v-SNAREs. <t>SDS-PAGE</t> analysis of in vitro binding of coatomer (top) and Arf1ΔN17p-Q71L (bottom) to GST (lanes 1 and 2) or GST fusions to v-SNAREs (lanes 3 and 4, Bet1p-GST; lanes 5 and 6, Sec22p-GST; lanes 7 and 8, GST-Bos1p). GST or GST fusion proteins were immobilized onto glutathione-agarose. Where indicated, 20 nM Glo3p (A) or Gcs1p (B) were added to the reaction, and 50% of the amount added is shown in lane 9. The guanine nucleotide on Arf1ΔN17p-Q71L was exchanged to GTP before the binding reaction, and 1.2 μM preexchanged Arf1ΔN17p-Q71L was added to the binding reaction. All reactions contained GTP. The coatomer concentration was 40 nM in the assay. After the binding reaction, the unbound proteins were removed by centrifugation. The proteins immobilized on the glutathione-agarose were separated by SDS-PAGE followed by <t>SyproRed</t> staining and analysis using the red fluorescent mode of a Storm PhosphorImager. In A, lanes 10 and 11 represent 20% of Arf1ΔN17p-Q71L and 50% of coatomer present in the reaction, respectively. In Bet1p-GST and Sec22p-GST, GST is the COOH-terminal fusion partner, whereas in GST-Bos1p it is in the NH 2 -terminal position.
    Sds Gel Electrophoresis, supplied by Abcam, used in various techniques. Bioz Stars score: 88/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    TaKaRa sds gel electrophoresis
    ARF-GAPs mediate Arf1 Δ N17p-Q71L and coatomer binding to the cytosolic domains of v-SNAREs. <t>SDS-PAGE</t> analysis of in vitro binding of coatomer (top) and Arf1ΔN17p-Q71L (bottom) to GST (lanes 1 and 2) or GST fusions to v-SNAREs (lanes 3 and 4, Bet1p-GST; lanes 5 and 6, Sec22p-GST; lanes 7 and 8, GST-Bos1p). GST or GST fusion proteins were immobilized onto glutathione-agarose. Where indicated, 20 nM Glo3p (A) or Gcs1p (B) were added to the reaction, and 50% of the amount added is shown in lane 9. The guanine nucleotide on Arf1ΔN17p-Q71L was exchanged to GTP before the binding reaction, and 1.2 μM preexchanged Arf1ΔN17p-Q71L was added to the binding reaction. All reactions contained GTP. The coatomer concentration was 40 nM in the assay. After the binding reaction, the unbound proteins were removed by centrifugation. The proteins immobilized on the glutathione-agarose were separated by SDS-PAGE followed by <t>SyproRed</t> staining and analysis using the red fluorescent mode of a Storm PhosphorImager. In A, lanes 10 and 11 represent 20% of Arf1ΔN17p-Q71L and 50% of coatomer present in the reaction, respectively. In Bet1p-GST and Sec22p-GST, GST is the COOH-terminal fusion partner, whereas in GST-Bos1p it is in the NH 2 -terminal position.
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    Agrisera sds gel electrophoresis
    ARF-GAPs mediate Arf1 Δ N17p-Q71L and coatomer binding to the cytosolic domains of v-SNAREs. <t>SDS-PAGE</t> analysis of in vitro binding of coatomer (top) and Arf1ΔN17p-Q71L (bottom) to GST (lanes 1 and 2) or GST fusions to v-SNAREs (lanes 3 and 4, Bet1p-GST; lanes 5 and 6, Sec22p-GST; lanes 7 and 8, GST-Bos1p). GST or GST fusion proteins were immobilized onto glutathione-agarose. Where indicated, 20 nM Glo3p (A) or Gcs1p (B) were added to the reaction, and 50% of the amount added is shown in lane 9. The guanine nucleotide on Arf1ΔN17p-Q71L was exchanged to GTP before the binding reaction, and 1.2 μM preexchanged Arf1ΔN17p-Q71L was added to the binding reaction. All reactions contained GTP. The coatomer concentration was 40 nM in the assay. After the binding reaction, the unbound proteins were removed by centrifugation. The proteins immobilized on the glutathione-agarose were separated by SDS-PAGE followed by <t>SyproRed</t> staining and analysis using the red fluorescent mode of a Storm PhosphorImager. In A, lanes 10 and 11 represent 20% of Arf1ΔN17p-Q71L and 50% of coatomer present in the reaction, respectively. In Bet1p-GST and Sec22p-GST, GST is the COOH-terminal fusion partner, whereas in GST-Bos1p it is in the NH 2 -terminal position.
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    Enzo Biochem sds gel electrophoresis
    ARF-GAPs mediate Arf1 Δ N17p-Q71L and coatomer binding to the cytosolic domains of v-SNAREs. <t>SDS-PAGE</t> analysis of in vitro binding of coatomer (top) and Arf1ΔN17p-Q71L (bottom) to GST (lanes 1 and 2) or GST fusions to v-SNAREs (lanes 3 and 4, Bet1p-GST; lanes 5 and 6, Sec22p-GST; lanes 7 and 8, GST-Bos1p). GST or GST fusion proteins were immobilized onto glutathione-agarose. Where indicated, 20 nM Glo3p (A) or Gcs1p (B) were added to the reaction, and 50% of the amount added is shown in lane 9. The guanine nucleotide on Arf1ΔN17p-Q71L was exchanged to GTP before the binding reaction, and 1.2 μM preexchanged Arf1ΔN17p-Q71L was added to the binding reaction. All reactions contained GTP. The coatomer concentration was 40 nM in the assay. After the binding reaction, the unbound proteins were removed by centrifugation. The proteins immobilized on the glutathione-agarose were separated by SDS-PAGE followed by <t>SyproRed</t> staining and analysis using the red fluorescent mode of a Storm PhosphorImager. In A, lanes 10 and 11 represent 20% of Arf1ΔN17p-Q71L and 50% of coatomer present in the reaction, respectively. In Bet1p-GST and Sec22p-GST, GST is the COOH-terminal fusion partner, whereas in GST-Bos1p it is in the NH 2 -terminal position.
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    GE Healthcare sds polyacrylamide gel electrophoresis page gel
    ARF-GAPs mediate Arf1 Δ N17p-Q71L and coatomer binding to the cytosolic domains of v-SNAREs. <t>SDS-PAGE</t> analysis of in vitro binding of coatomer (top) and Arf1ΔN17p-Q71L (bottom) to GST (lanes 1 and 2) or GST fusions to v-SNAREs (lanes 3 and 4, Bet1p-GST; lanes 5 and 6, Sec22p-GST; lanes 7 and 8, GST-Bos1p). GST or GST fusion proteins were immobilized onto glutathione-agarose. Where indicated, 20 nM Glo3p (A) or Gcs1p (B) were added to the reaction, and 50% of the amount added is shown in lane 9. The guanine nucleotide on Arf1ΔN17p-Q71L was exchanged to GTP before the binding reaction, and 1.2 μM preexchanged Arf1ΔN17p-Q71L was added to the binding reaction. All reactions contained GTP. The coatomer concentration was 40 nM in the assay. After the binding reaction, the unbound proteins were removed by centrifugation. The proteins immobilized on the glutathione-agarose were separated by SDS-PAGE followed by <t>SyproRed</t> staining and analysis using the red fluorescent mode of a Storm PhosphorImager. In A, lanes 10 and 11 represent 20% of Arf1ΔN17p-Q71L and 50% of coatomer present in the reaction, respectively. In Bet1p-GST and Sec22p-GST, GST is the COOH-terminal fusion partner, whereas in GST-Bos1p it is in the NH 2 -terminal position.
    Sds Polyacrylamide Gel Electrophoresis Page Gel, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 91/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher sds polyarcylamide gel electrophoresis gel
    ARF-GAPs mediate Arf1 Δ N17p-Q71L and coatomer binding to the cytosolic domains of v-SNAREs. <t>SDS-PAGE</t> analysis of in vitro binding of coatomer (top) and Arf1ΔN17p-Q71L (bottom) to GST (lanes 1 and 2) or GST fusions to v-SNAREs (lanes 3 and 4, Bet1p-GST; lanes 5 and 6, Sec22p-GST; lanes 7 and 8, GST-Bos1p). GST or GST fusion proteins were immobilized onto glutathione-agarose. Where indicated, 20 nM Glo3p (A) or Gcs1p (B) were added to the reaction, and 50% of the amount added is shown in lane 9. The guanine nucleotide on Arf1ΔN17p-Q71L was exchanged to GTP before the binding reaction, and 1.2 μM preexchanged Arf1ΔN17p-Q71L was added to the binding reaction. All reactions contained GTP. The coatomer concentration was 40 nM in the assay. After the binding reaction, the unbound proteins were removed by centrifugation. The proteins immobilized on the glutathione-agarose were separated by SDS-PAGE followed by <t>SyproRed</t> staining and analysis using the red fluorescent mode of a Storm PhosphorImager. In A, lanes 10 and 11 represent 20% of Arf1ΔN17p-Q71L and 50% of coatomer present in the reaction, respectively. In Bet1p-GST and Sec22p-GST, GST is the COOH-terminal fusion partner, whereas in GST-Bos1p it is in the NH 2 -terminal position.
    Sds Polyarcylamide Gel Electrophoresis Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 78/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher sds polyacrylamide gel electrophoresis page gel
    Envelope glycoprotein incorporation into virions. Virions were pelleted, lysed, separated by <t>SDS-PAGE,</t> and visualized by Western blotting. (A) Virion-associated gp120 was detected using anti-gp120 MAb 3.11H, and p27 capsid protein was detected by MAb 2F12. (B) Densitometric analysis [(variant gp120/p27)/(parental gp120/p27)] of bands in panel A.
    Sds Polyacrylamide Gel Electrophoresis Page Gel, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Covance sds polyacrylamide gel electrophoresis page gel
    Envelope glycoprotein incorporation into virions. Virions were pelleted, lysed, separated by <t>SDS-PAGE,</t> and visualized by Western blotting. (A) Virion-associated gp120 was detected using anti-gp120 MAb 3.11H, and p27 capsid protein was detected by MAb 2F12. (B) Densitometric analysis [(variant gp120/p27)/(parental gp120/p27)] of bands in panel A.
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    GE Healthcare sds gel electrophoresis calibration kit
    Envelope glycoprotein incorporation into virions. Virions were pelleted, lysed, separated by <t>SDS-PAGE,</t> and visualized by Western blotting. (A) Virion-associated gp120 was detected using anti-gp120 MAb 3.11H, and p27 capsid protein was detected by MAb 2F12. (B) Densitometric analysis [(variant gp120/p27)/(parental gp120/p27)] of bands in panel A.
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    Thermo Fisher sds polyacrylamide gradient gel electrophoresis
    Envelope glycoprotein incorporation into virions. Virions were pelleted, lysed, separated by <t>SDS-PAGE,</t> and visualized by Western blotting. (A) Virion-associated gp120 was detected using anti-gp120 MAb 3.11H, and p27 capsid protein was detected by MAb 2F12. (B) Densitometric analysis [(variant gp120/p27)/(parental gp120/p27)] of bands in panel A.
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    Bio-Rad sds polyacrylamide gradient gel electrophoresis
    Envelope glycoprotein incorporation into virions. Virions were pelleted, lysed, separated by <t>SDS-PAGE,</t> and visualized by Western blotting. (A) Virion-associated gp120 was detected using anti-gp120 MAb 3.11H, and p27 capsid protein was detected by MAb 2F12. (B) Densitometric analysis [(variant gp120/p27)/(parental gp120/p27)] of bands in panel A.
    Sds Polyacrylamide Gradient Gel Electrophoresis, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 91/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad sds page electrophoresis aliquots
    The effect of o -methylated 3HPs on: i) the HEWL fibril formation, ii) its α-to-β conformational rearrangement, iii) the HEWL acid-induced proteolysis, and iv) the HEWL thermal stability. (A–B) AFM micrographs of HEWL solutions (0.2 mM) previously incubated during 16d at pH 2.0 and 60 °C either (A) in buffer, or (B) in the presence of 20 mM 2 m-3HP. The scale bar represents 0.5 μm. (C-D) Far-UV CD spectra of HEWL incubated during 0, 7 and 17 days at pH 2.0 and 60 °C either (C) in buffer, or (D) in the presence of 20 mM 2 m-3HP. (E) <t>SDS-PAGE</t> gel analysis of solutions containing HEWL incubated at pH 2.0 and 60 °C during 0 and 10d either in the absence or in the presence of different o -methylated 3HPs. (F) Differential scanning calorimetry thermograms of different HEWL solutions (0.2 mM) at pH 2.0 acquired in the absence or in the presence of 20 mM of the different o -methylated 3HPs.
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    Image Search Results


    TCFβ1 is phosphorylated in vitro in an activation-dependent manner. Full-length TCFβ1 (residues 1 to 301) expressed as a GST fusion protein was incubated with cell extracts from Jurkat cells activated with PMA (5 ng/ml), PMA (5 ng/ml) plus PHA (5 μg/ml), or UV for the indicated lengths of time, processed for in vitro kinase assay, analyzed by SDS-PAGE, and visualized by autoradiography. GST alone was used as a negative control.

    Journal: Molecular and Cellular Biology

    Article Title: Jun Kinase Phosphorylates and Regulates the DNA Binding Activity of an Octamer Binding Protein, T-Cell Factor ?1 †

    doi:

    Figure Lengend Snippet: TCFβ1 is phosphorylated in vitro in an activation-dependent manner. Full-length TCFβ1 (residues 1 to 301) expressed as a GST fusion protein was incubated with cell extracts from Jurkat cells activated with PMA (5 ng/ml), PMA (5 ng/ml) plus PHA (5 μg/ml), or UV for the indicated lengths of time, processed for in vitro kinase assay, analyzed by SDS-PAGE, and visualized by autoradiography. GST alone was used as a negative control.

    Article Snippet: Kinase reactions were carried out at 30°C for 15 min and stopped by washing once with kinase buffer; proteins eluted with 2× sodium dodecyl sulfate (SDS) sample buffer (60 mM Tris [pH 6.8], 2.3% SDS, 10% glycerol, 5% β-mercaptoethanol), resolved by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to Immobilon membranes (Millipore, Bedford, Mass.), and subjected to autoradiography.

    Techniques: In Vitro, Activation Assay, Incubation, Kinase Assay, SDS Page, Autoradiography, Negative Control

    TCFβ1 is phosphorylated in vivo by serine/threonine kinases. (A) Phosphorylation of epitope-tagged TCFβ1 in Jurkat cells after activation. Jurkat cells that were transfected with pJF-HA vector alone, or the same vector expressing full-length TCFβ1, and then metabolically labelled ( 32 P) were activated with PMA (5 ng/ml) plus PHA (5 μg/ml). TCFβ1 was immunoprecipitated from Jurkat cell lysates with anti-HA antibody, resolved on SDS-PAGE gels, transferred to Immobilon membranes, and visualized by autoradiography. The arrow indicates the TCFβ1 band. (B) Expression of epitope-tagged TCFβ1 in Jurkat cells after transient transfection. Jurkat cells were transfected, and 48 h later, expression of HA-TCFβ1 protein was determined by Western immunoblotting. TCFβ1 was immunoprecipitated from Jurkat cell lysates with anti-HA antibody (12CA5), resolved on SDS-polyacrylamide gels, and Western blot probed with anti-HA antibody. The western immunoblot shows that the levels of expression of HA-TCFβ1 in activated and unactivated Jurkat cells are similar, as evident in panel A. The arrow indicates the TCFβ1 band. (C) PAA analysis of full-length TCFβ1, labelled in vivo, from activated Jurkat cells. Phosphoserine and some phosphothreonine were detectable, but phosphotyrosine was not.

    Journal: Molecular and Cellular Biology

    Article Title: Jun Kinase Phosphorylates and Regulates the DNA Binding Activity of an Octamer Binding Protein, T-Cell Factor ?1 †

    doi:

    Figure Lengend Snippet: TCFβ1 is phosphorylated in vivo by serine/threonine kinases. (A) Phosphorylation of epitope-tagged TCFβ1 in Jurkat cells after activation. Jurkat cells that were transfected with pJF-HA vector alone, or the same vector expressing full-length TCFβ1, and then metabolically labelled ( 32 P) were activated with PMA (5 ng/ml) plus PHA (5 μg/ml). TCFβ1 was immunoprecipitated from Jurkat cell lysates with anti-HA antibody, resolved on SDS-PAGE gels, transferred to Immobilon membranes, and visualized by autoradiography. The arrow indicates the TCFβ1 band. (B) Expression of epitope-tagged TCFβ1 in Jurkat cells after transient transfection. Jurkat cells were transfected, and 48 h later, expression of HA-TCFβ1 protein was determined by Western immunoblotting. TCFβ1 was immunoprecipitated from Jurkat cell lysates with anti-HA antibody (12CA5), resolved on SDS-polyacrylamide gels, and Western blot probed with anti-HA antibody. The western immunoblot shows that the levels of expression of HA-TCFβ1 in activated and unactivated Jurkat cells are similar, as evident in panel A. The arrow indicates the TCFβ1 band. (C) PAA analysis of full-length TCFβ1, labelled in vivo, from activated Jurkat cells. Phosphoserine and some phosphothreonine were detectable, but phosphotyrosine was not.

    Article Snippet: Kinase reactions were carried out at 30°C for 15 min and stopped by washing once with kinase buffer; proteins eluted with 2× sodium dodecyl sulfate (SDS) sample buffer (60 mM Tris [pH 6.8], 2.3% SDS, 10% glycerol, 5% β-mercaptoethanol), resolved by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to Immobilon membranes (Millipore, Bedford, Mass.), and subjected to autoradiography.

    Techniques: In Vivo, Activation Assay, Transfection, Plasmid Preparation, Expressing, Metabolic Labelling, Immunoprecipitation, SDS Page, Autoradiography, Western Blot

    Bak undergoes tyrosine dephosphorylated during the initiation of apoptosis. ( A ) 2D gel analysis of Bak from HT1080 cells using narrow-range linear pI gradient IPG-focusing strips and 15% Tris-glycine SDS–PAGE showed partial dephosphorylation after UV treatment, complete dephosphorylation was achieved by λ-phosphatase treatment. ( B ) 2D gel analysis of Bak of mitochondrial extracts from HT1080 cells treated either with ser/thr (PP1) or tyr (YOP) phosphatase, which resulted in new Bak species (arrowed). ( C ) Histograms of FACS analysis of Bak Ab-1-specific fluorescence in control untreated HT1080 cells, HT1080 cells±pre-treatment for 30 min before UV treatment with phophatase inhibitors sodium stibogluconate (SS; 110 μM), phenylarsine oxide (PAO; 5 μM), Cyclosporin A (75 μM) and Calyculin A (2 nM). Samples were analysed 4 h after UV damage. ( D ) Quantification of the increase in Bak Ab-1-specific fluorescence±4 h apoptotic stimuli camptothecin (CPT; 6 μM), Etoposide (EP; 10 μM), Staurosporine (STS; 100 nM) or 5 mJ/cm 2 UV in the presence and absence of tyrosine phoshatase inhibitors 110 μM SS or 5 μM PAO with the y -axis showing levels of Bak-specific fluorescence and the x -axis showing treatment conditions.

    Journal: The EMBO Journal

    Article Title: Tyrosine dephosphorylation is required for Bak activation in apoptosis

    doi: 10.1038/emboj.2010.244

    Figure Lengend Snippet: Bak undergoes tyrosine dephosphorylated during the initiation of apoptosis. ( A ) 2D gel analysis of Bak from HT1080 cells using narrow-range linear pI gradient IPG-focusing strips and 15% Tris-glycine SDS–PAGE showed partial dephosphorylation after UV treatment, complete dephosphorylation was achieved by λ-phosphatase treatment. ( B ) 2D gel analysis of Bak of mitochondrial extracts from HT1080 cells treated either with ser/thr (PP1) or tyr (YOP) phosphatase, which resulted in new Bak species (arrowed). ( C ) Histograms of FACS analysis of Bak Ab-1-specific fluorescence in control untreated HT1080 cells, HT1080 cells±pre-treatment for 30 min before UV treatment with phophatase inhibitors sodium stibogluconate (SS; 110 μM), phenylarsine oxide (PAO; 5 μM), Cyclosporin A (75 μM) and Calyculin A (2 nM). Samples were analysed 4 h after UV damage. ( D ) Quantification of the increase in Bak Ab-1-specific fluorescence±4 h apoptotic stimuli camptothecin (CPT; 6 μM), Etoposide (EP; 10 μM), Staurosporine (STS; 100 nM) or 5 mJ/cm 2 UV in the presence and absence of tyrosine phoshatase inhibitors 110 μM SS or 5 μM PAO with the y -axis showing levels of Bak-specific fluorescence and the x -axis showing treatment conditions.

    Article Snippet: Bound proteins were eluted in denaturing SDS buffer and electrophoresed on 4–12% NuPAGE bis-Tris gels (Invitrogen).

    Techniques: Two-Dimensional Gel Electrophoresis, SDS Page, De-Phosphorylation Assay, FACS, Fluorescence, Cycling Probe Technology

    Phosphor imager picture of SDS-PAGE gel of the product from the 125 I-cetuximab labelling reaction. The 125 I-cetuximab labelling reaction with 73.1, 112.2 and 150.0 MBq with miniaturized IODOGEN-coated mAb method demonstrating unaffected integrity with respect to the molecular weight. Conditions: 50 μg (0.33 nmol) cetuximab, 2.5 μg (5.7 nmol) IODOGEN, 250 μL reaction volume, 90-s reaction time.

    Journal: EJNMMI Research

    Article Title: The ultimate radiochemical nightmare: upon radio-iodination of Botulinum neurotoxin A, the introduced iodine atom itself seems to be fatal for the bioactivity of this macromolecule

    doi: 10.1186/s13550-015-0083-5

    Figure Lengend Snippet: Phosphor imager picture of SDS-PAGE gel of the product from the 125 I-cetuximab labelling reaction. The 125 I-cetuximab labelling reaction with 73.1, 112.2 and 150.0 MBq with miniaturized IODOGEN-coated mAb method demonstrating unaffected integrity with respect to the molecular weight. Conditions: 50 μg (0.33 nmol) cetuximab, 2.5 μg (5.7 nmol) IODOGEN, 250 μL reaction volume, 90-s reaction time.

    Article Snippet: Gel electrophoresis was performed on a Pharmacia Phastgel System using 7.5% SDS-PAGE gel electrophoresis gels (Amersham Biosciences, Roosendaal, The Netherlands) under non-reducing conditions and analyzed on a phosphor imager (Molecular Dynamics, Zoetermeer, The Netherlands) and quantified with ImageQuant software.

    Techniques: SDS Page, Molecular Weight

    Aldosterone (ALD) increases activity levels and protein expression of Na + -K + -2Cl − cotransport protein (NKCC1). A : NKCC1 activity was determined through 86 Rb uptake, which was measured in an isosmotic solution containing 1 mM ouabain, at 2, 4, 8, and 12 h after ALD administration. B : HT-29 cells were treated with ALD for the times and doses indicated and lysed by RIPA buffer with cocktail protein inhibitors; proteins were resolved by SDS-PAGE. Western blot was probed sequentially with antibodies to NKCC1 and β-actin as the control. ALD treatment increases NKCC1 protein expression. The time frame of upregulation is from 2 h up to 24 h after application of ALD (1 μM). C : further investigation revealed that the induction of NKCC1 by ALD (1 μM) starts 30–40 min after treatment. D : immunoblot of NKCC1 protein expression for different doses of ALD. The threshold for an effective dose of ALD on NKCC1 protein expression is on the order of only 10 pM. Bar graph results are means ± SD from 3 independent experiments, the ordinate represents relative expression, defined as expression level relative to the time 0 or dosage point. E : increase in NKCC1 protein levels is mediated via the activation of mineralocorticoid receptors (MR). HT-29 cells were treated with or without a specific inhibitor of MR, eplerenone (20 μM), and then applied with 1 μM ALD for the indicated times. NKCC1 protein expression was detected in cell lysates by a Western blot, using antibodies to NKCC1, and β-actin as a loading control. Open bar: control, nontreated sample; the vehicle was 100% alcohol, in which the ALD and eplerenone were dissolved. Statistical significance: **** P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Direct control of Na+-K+-2Cl−-cotransport protein (NKCC1) expression with aldosterone

    doi: 10.1152/ajpcell.00096.2013

    Figure Lengend Snippet: Aldosterone (ALD) increases activity levels and protein expression of Na + -K + -2Cl − cotransport protein (NKCC1). A : NKCC1 activity was determined through 86 Rb uptake, which was measured in an isosmotic solution containing 1 mM ouabain, at 2, 4, 8, and 12 h after ALD administration. B : HT-29 cells were treated with ALD for the times and doses indicated and lysed by RIPA buffer with cocktail protein inhibitors; proteins were resolved by SDS-PAGE. Western blot was probed sequentially with antibodies to NKCC1 and β-actin as the control. ALD treatment increases NKCC1 protein expression. The time frame of upregulation is from 2 h up to 24 h after application of ALD (1 μM). C : further investigation revealed that the induction of NKCC1 by ALD (1 μM) starts 30–40 min after treatment. D : immunoblot of NKCC1 protein expression for different doses of ALD. The threshold for an effective dose of ALD on NKCC1 protein expression is on the order of only 10 pM. Bar graph results are means ± SD from 3 independent experiments, the ordinate represents relative expression, defined as expression level relative to the time 0 or dosage point. E : increase in NKCC1 protein levels is mediated via the activation of mineralocorticoid receptors (MR). HT-29 cells were treated with or without a specific inhibitor of MR, eplerenone (20 μM), and then applied with 1 μM ALD for the indicated times. NKCC1 protein expression was detected in cell lysates by a Western blot, using antibodies to NKCC1, and β-actin as a loading control. Open bar: control, nontreated sample; the vehicle was 100% alcohol, in which the ALD and eplerenone were dissolved. Statistical significance: **** P

    Article Snippet: Proteins were fractionated by SDS-PAGE gel electrophoresis and transferred to a PVDF blotting membrane (Whatman, Piscataway, NJ).

    Techniques: Activity Assay, Expressing, SDS Page, Western Blot, Activation Assay

    Protein profile of the CLDs and MFGMs by 1D SDS–PAGE. CLDs were prepared from mammary acini purified from mouse mammary gland at day 10 of lactation, and MFGMs were recovered from MFGs isolated from mouse milk collected at the same time point, as described in Materials and Methods . Proteins (∼8 μg) were loaded on 4–12% polyacrylamide gels (NuPAGE Novex, 4–12% Bis-Tris gel, NP002) in MES buffer and further stained with SimplyBlue SafeStain G250 (LC6060; Invitrogen). The relative molecular masses (kilodaltons) are indicated. Marker proteins appeared specifically enriched in each preparation: PLIN2 in CLDs, and BTN1 and MFG-E8 in MFGMs.

    Journal: Molecular Biology of the Cell

    Article Title: The endoplasmic reticulum and casein-containing vesicles contribute to milk fat globule membrane

    doi: 10.1091/mbc.E16-06-0364

    Figure Lengend Snippet: Protein profile of the CLDs and MFGMs by 1D SDS–PAGE. CLDs were prepared from mammary acini purified from mouse mammary gland at day 10 of lactation, and MFGMs were recovered from MFGs isolated from mouse milk collected at the same time point, as described in Materials and Methods . Proteins (∼8 μg) were loaded on 4–12% polyacrylamide gels (NuPAGE Novex, 4–12% Bis-Tris gel, NP002) in MES buffer and further stained with SimplyBlue SafeStain G250 (LC6060; Invitrogen). The relative molecular masses (kilodaltons) are indicated. Marker proteins appeared specifically enriched in each preparation: PLIN2 in CLDs, and BTN1 and MFG-E8 in MFGMs.

    Article Snippet: Western blot A 20-μg amount of total protein from the MFGM fractions was analyzed by SDS-polyacrylamide 12% gel electrophoresis (SDS–PAGE) and transferred onto Hybond nitrocellulose membrane (Amersham).

    Techniques: SDS Page, Purification, Isolation, Staining, Marker

    Experimental flowchart for the identification of the proteins associated with murine MESC CLDs and with MFGMs. MFGs were isolated from mouse milk (L10), and MFGM proteins were extracted. MESCs (acini) were purified from mouse mammary glands collected at L10 and homogenized, and CLDs were purified by flotation on a sucrose gradient. Proteins from both CLDs and MFGMs were separated by 1D SDS–PAGE. The gel lanes were sliced into 26 pieces, the proteins were subjected to in-gel trypsin digestion, and the hydrolysates were analyzed by LC-MS/MS. The proteins identified by at least two peptides were classified for both function and cellular localization based on their GO terms (Uniprot KB database).

    Journal: Molecular Biology of the Cell

    Article Title: The endoplasmic reticulum and casein-containing vesicles contribute to milk fat globule membrane

    doi: 10.1091/mbc.E16-06-0364

    Figure Lengend Snippet: Experimental flowchart for the identification of the proteins associated with murine MESC CLDs and with MFGMs. MFGs were isolated from mouse milk (L10), and MFGM proteins were extracted. MESCs (acini) were purified from mouse mammary glands collected at L10 and homogenized, and CLDs were purified by flotation on a sucrose gradient. Proteins from both CLDs and MFGMs were separated by 1D SDS–PAGE. The gel lanes were sliced into 26 pieces, the proteins were subjected to in-gel trypsin digestion, and the hydrolysates were analyzed by LC-MS/MS. The proteins identified by at least two peptides were classified for both function and cellular localization based on their GO terms (Uniprot KB database).

    Article Snippet: Western blot A 20-μg amount of total protein from the MFGM fractions was analyzed by SDS-polyacrylamide 12% gel electrophoresis (SDS–PAGE) and transferred onto Hybond nitrocellulose membrane (Amersham).

    Techniques: Isolation, Purification, SDS Page, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    Analysis of the different mouse milk fractions and quality of the MFGMs. (A) Whole mouse milk as well as the milk fractions corresponding to caseins, lactoserum, and MFGMs were analyzed by SDS–PAGE on 12.5% acrylamide gel. Caseins appeared to be the main proteins present in mouse milk and were only faintly present in the lactoserum and MFGs fractions. The specific protein pattern observed for MFGMs indicated the minimal contamination by caseins or lactoserum proteins, as well as the enrichment of some proteins in this fraction. Relative molecular masses (kilodaltons) and casein isoforms are indicated on the left. (B) MFGs purified from mouse milk and isolated MFGMs were analyzed by transmission electron microscopy after negative coloration. The MFGs appeared as round structures with a lipid core, whereas MFGMs were devoid of neutral lipid core, cellular debris, or membranous organelles.

    Journal: Molecular Biology of the Cell

    Article Title: The endoplasmic reticulum and casein-containing vesicles contribute to milk fat globule membrane

    doi: 10.1091/mbc.E16-06-0364

    Figure Lengend Snippet: Analysis of the different mouse milk fractions and quality of the MFGMs. (A) Whole mouse milk as well as the milk fractions corresponding to caseins, lactoserum, and MFGMs were analyzed by SDS–PAGE on 12.5% acrylamide gel. Caseins appeared to be the main proteins present in mouse milk and were only faintly present in the lactoserum and MFGs fractions. The specific protein pattern observed for MFGMs indicated the minimal contamination by caseins or lactoserum proteins, as well as the enrichment of some proteins in this fraction. Relative molecular masses (kilodaltons) and casein isoforms are indicated on the left. (B) MFGs purified from mouse milk and isolated MFGMs were analyzed by transmission electron microscopy after negative coloration. The MFGs appeared as round structures with a lipid core, whereas MFGMs were devoid of neutral lipid core, cellular debris, or membranous organelles.

    Article Snippet: Western blot A 20-μg amount of total protein from the MFGM fractions was analyzed by SDS-polyacrylamide 12% gel electrophoresis (SDS–PAGE) and transferred onto Hybond nitrocellulose membrane (Amersham).

    Techniques: SDS Page, Acrylamide Gel Assay, Purification, Isolation, Transmission Assay, Electron Microscopy

    Isolation and quality of the purified CLDs from mouse lactating mammary gland. (A) Mammary acini purified from mouse mammary gland at day 10 of lactation were washed and homogenized. Total lysate (T) was centrifuged at 800 × g for 10 min at 4°C. The postnuclear supernatant (S1) was subsequently centrifuged at 110,000 × g for 1 h at 4°C to isolate cellular membrane (P2) and soluble material (S2). After centrifugation of the total lysate at 274,000 × g for 1 h at 4°C, the top white layer was the CLD fraction. (B) Isolated CLDs were analyzed by differential interference contrast microscopy (a; nt, not treated) and fluorescence microscopy after BODIPY 493/503 staining (lipids; b, e, h, k). CLDs were counterstained with Alexa Fluor 594–conjugated WGA (d), rhodamine-conjugated phalloidin (actin; g), or Alexa 594–conjugated CTxB (GM1; j) and merged (c, f, i, l) in order to visualize potential contaminations. Scale bar, 10 μm. (C) Proteins extracted from the different fractions were separated by SDS–PAGE and stained with Coomassie blue. Note the distinct banding pattern of CLDs. (D) The same protein samples were also subjected to Western blotting to test for contamination from other cellular fractions. Specific antibodies were used to probe for marker proteins of different cellular organelles/fractions: PLIN2 (CLD protein), BTN1 (MFG protein), E-cadherin (PM), β-actin (cytosol), PdiA3 and GRP78 (ER lumen), calnexin and Stx-18 (ER membrane), and GM130 (Golgi). Note the strong enrichment of PLIN2 in the CLD fraction. BTN1, butyrophilin; E-Cad, E-cadherin; GM130, Golgi matrix protein 130; GRP78, glucose-regulated protein 78; M, whole milk; P1, pellet 1; P2, pellet 2; PdiA3, protein disulfide isomerase A3; PLIN2, perilipin2; S1, supernatant 1; S2, supernatant 2; Stx-18, syntaxin 18; T, total extract.

    Journal: Molecular Biology of the Cell

    Article Title: The endoplasmic reticulum and casein-containing vesicles contribute to milk fat globule membrane

    doi: 10.1091/mbc.E16-06-0364

    Figure Lengend Snippet: Isolation and quality of the purified CLDs from mouse lactating mammary gland. (A) Mammary acini purified from mouse mammary gland at day 10 of lactation were washed and homogenized. Total lysate (T) was centrifuged at 800 × g for 10 min at 4°C. The postnuclear supernatant (S1) was subsequently centrifuged at 110,000 × g for 1 h at 4°C to isolate cellular membrane (P2) and soluble material (S2). After centrifugation of the total lysate at 274,000 × g for 1 h at 4°C, the top white layer was the CLD fraction. (B) Isolated CLDs were analyzed by differential interference contrast microscopy (a; nt, not treated) and fluorescence microscopy after BODIPY 493/503 staining (lipids; b, e, h, k). CLDs were counterstained with Alexa Fluor 594–conjugated WGA (d), rhodamine-conjugated phalloidin (actin; g), or Alexa 594–conjugated CTxB (GM1; j) and merged (c, f, i, l) in order to visualize potential contaminations. Scale bar, 10 μm. (C) Proteins extracted from the different fractions were separated by SDS–PAGE and stained with Coomassie blue. Note the distinct banding pattern of CLDs. (D) The same protein samples were also subjected to Western blotting to test for contamination from other cellular fractions. Specific antibodies were used to probe for marker proteins of different cellular organelles/fractions: PLIN2 (CLD protein), BTN1 (MFG protein), E-cadherin (PM), β-actin (cytosol), PdiA3 and GRP78 (ER lumen), calnexin and Stx-18 (ER membrane), and GM130 (Golgi). Note the strong enrichment of PLIN2 in the CLD fraction. BTN1, butyrophilin; E-Cad, E-cadherin; GM130, Golgi matrix protein 130; GRP78, glucose-regulated protein 78; M, whole milk; P1, pellet 1; P2, pellet 2; PdiA3, protein disulfide isomerase A3; PLIN2, perilipin2; S1, supernatant 1; S2, supernatant 2; Stx-18, syntaxin 18; T, total extract.

    Article Snippet: Western blot A 20-μg amount of total protein from the MFGM fractions was analyzed by SDS-polyacrylamide 12% gel electrophoresis (SDS–PAGE) and transferred onto Hybond nitrocellulose membrane (Amersham).

    Techniques: Isolation, Purification, Centrifugation, Microscopy, Fluorescence, Staining, Whole Genome Amplification, SDS Page, Western Blot, Marker

    Analysis of ABCB4 and ABCG2 protein expression by Western blot A. Cell lysates from the placentas of female SD rats were electrophoretically separated by SDS-PAGE, transferred to PVDF membranes and probed using ABCB4 and ABCG2 antibodies. ABCB4 and ABCG2 levels were compared between the Cd-treated groups and the control group. B. Relative quantities based on OD are shown. Data are presented as means±SD of relative fold changes. *P

    Journal: Oncotarget

    Article Title: Down-regulation of ABCG2 and ABCB4 transporters in the placenta of rats exposed to cadmium

    doi: 10.18632/oncotarget.9415

    Figure Lengend Snippet: Analysis of ABCB4 and ABCG2 protein expression by Western blot A. Cell lysates from the placentas of female SD rats were electrophoretically separated by SDS-PAGE, transferred to PVDF membranes and probed using ABCB4 and ABCG2 antibodies. ABCB4 and ABCG2 levels were compared between the Cd-treated groups and the control group. B. Relative quantities based on OD are shown. Data are presented as means±SD of relative fold changes. *P

    Article Snippet: The strips were equilibrated by agitation for 30 min, loaded onto a 12.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and run on an Ettan™ DALT Six System (GE Healthcare).

    Techniques: Expressing, Western Blot, SDS Page

    MALDI-TOF spectrum of Bovine SP-B dimer sample eluted from SDS gel of ETS-exposed surfactant sample. The ABI Voyager-DE STR MALDI-TOF was run in linear mode with delayed extraction and negative polarity. The instrument had an accelerating voltage of 2000

    Journal:

    Article Title: Environmental Tobacco Smoke Effects on Lung Surfactant Film Organization

    doi: 10.1016/j.bbamem.2008.11.021

    Figure Lengend Snippet: MALDI-TOF spectrum of Bovine SP-B dimer sample eluted from SDS gel of ETS-exposed surfactant sample. The ABI Voyager-DE STR MALDI-TOF was run in linear mode with delayed extraction and negative polarity. The instrument had an accelerating voltage of 2000

    Article Snippet: The eluted protein fractions were then identified by SDS-PAGE gel electrophoresis (10–20% Tricine Gel, Invitrogen, Carlsbad, CA).

    Techniques: SDS-Gel

    SDS-PAGE and Western blot analysis of a serum sample (left) and milk sample (right), as indicated. The serum sample was diluted 1:1600 and the milk sample was diluted 1:40. In both cases, the purified standard mink IgG preparation was also included. All samples were electrophoresed under reducing conditions on 12% NuPAGE Bis–Tris gel as described in “ Methods ” section. For Western blotting (WB), the blot was developed with polyclonal goat anti-ferret IgG, followed by alkaline phosphatase-conjugated rabbit anti-goat antibody (see “ Methods ” section). SDS-PAGE: Lane 1: molecular weight marker; Lane 2: serum/milk sample; Lane 3: purified standard mink IgG. WB: Lane 4: molecular weight marker; Lane 5: serum/milk sample; Lane 6: purified standard mink IgG. The positions of the molecular weight marker proteins (20–100 kDa) are indicated to the left of the gels and blots

    Journal: Acta Veterinaria Scandinavica

    Article Title: Quantitative immunoassay for mink immunoglobulin in serum and milk

    doi: 10.1186/s13028-018-0391-7

    Figure Lengend Snippet: SDS-PAGE and Western blot analysis of a serum sample (left) and milk sample (right), as indicated. The serum sample was diluted 1:1600 and the milk sample was diluted 1:40. In both cases, the purified standard mink IgG preparation was also included. All samples were electrophoresed under reducing conditions on 12% NuPAGE Bis–Tris gel as described in “ Methods ” section. For Western blotting (WB), the blot was developed with polyclonal goat anti-ferret IgG, followed by alkaline phosphatase-conjugated rabbit anti-goat antibody (see “ Methods ” section). SDS-PAGE: Lane 1: molecular weight marker; Lane 2: serum/milk sample; Lane 3: purified standard mink IgG. WB: Lane 4: molecular weight marker; Lane 5: serum/milk sample; Lane 6: purified standard mink IgG. The positions of the molecular weight marker proteins (20–100 kDa) are indicated to the left of the gels and blots

    Article Snippet: Eluted IgG fractions were pooled and dialyzed against PBS overnight at 4 °C and then analyzed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) (12% Bis–Tris NuPAGE, Life Technologies, Taastrup, Denmark) followed by silver staining to estimate purity ( > 90%, Fig. ).

    Techniques: SDS Page, Western Blot, Purification, Molecular Weight, Marker

    IRF-3 5D-inducible expression of ISG56 gene. rtTA-Neo (lanes 1 to 5) and rtTA-IRF-3 5D (lanes 6 to 10) Jurkat cells were exposed to DOX (1 μg/ml) and anti-IFN antibodies for the indicated times. Whole-cell extracts (50 μg) were subjected to SDS-PAGE and analyzed by immunoblotting with anti-ISG56 antibodies. Membranes were stripped and reprobed with anti-IRF-3 and anti-actin antibodies.

    Journal: Journal of Virology

    Article Title: Transcriptional Profiling of Interferon Regulatory Factor 3 Target Genes: Direct Involvement in the Regulation of Interferon-Stimulated Genes

    doi: 10.1128/JVI.76.11.5532-5539.2002

    Figure Lengend Snippet: IRF-3 5D-inducible expression of ISG56 gene. rtTA-Neo (lanes 1 to 5) and rtTA-IRF-3 5D (lanes 6 to 10) Jurkat cells were exposed to DOX (1 μg/ml) and anti-IFN antibodies for the indicated times. Whole-cell extracts (50 μg) were subjected to SDS-PAGE and analyzed by immunoblotting with anti-ISG56 antibodies. Membranes were stripped and reprobed with anti-IRF-3 and anti-actin antibodies.

    Article Snippet: Whole-cell extracts (50 μg) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred on nitrocellulose membrane (Bio-Rad).

    Techniques: Expressing, SDS Page

    Immunoblot of sialome proteins from Gmm , Gpd , Gff and Gpg . Same amount of sialome proteins from Gmm , Gpd , Gff and Gpg were probed with (A) rec- Gmm Tsal1, (B) rec- Gmm TSGF-1 and (C) rec- Gmm TSGF-2 antibodies, respectively. (D) Gpg sialome components, equivalent to 0.2 pairs of salivary glands, are analyzed on Coomassie Blue stained SDS-PAGE (lane 2), and by immunoblot analysis using rec- Gmm TSGF-2 antibodies (lane 3) and anti- Gff saliva antibodies (lane 4). Lane 1 shows molecular marker. * indicates TSGF-2 corresponding protein band.

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Immunogenicity and Serological Cross-Reactivity of Saliva Proteins among Different Tsetse Species

    doi: 10.1371/journal.pntd.0004038

    Figure Lengend Snippet: Immunoblot of sialome proteins from Gmm , Gpd , Gff and Gpg . Same amount of sialome proteins from Gmm , Gpd , Gff and Gpg were probed with (A) rec- Gmm Tsal1, (B) rec- Gmm TSGF-1 and (C) rec- Gmm TSGF-2 antibodies, respectively. (D) Gpg sialome components, equivalent to 0.2 pairs of salivary glands, are analyzed on Coomassie Blue stained SDS-PAGE (lane 2), and by immunoblot analysis using rec- Gmm TSGF-2 antibodies (lane 3) and anti- Gff saliva antibodies (lane 4). Lane 1 shows molecular marker. * indicates TSGF-2 corresponding protein band.

    Article Snippet: SDS Polyacrylamide Gel Electrophoresis (PAGE) analysis and immunoblotting Same amount of total sialome proteins obtained from dissected SG (or extracts from the same number of dissected salivary glands) were analyzed by 12% SDS-PAGE under reducing conditions and either stained by coomasie blue, or transferred to nitrocellulose membranes (BioRad, Cat # 162–0112) according to standard protocols [ ].

    Techniques: Staining, SDS Page, Marker

    SDS PAGE analysis of saliva from different tsetse species. Lanes 1–4 show protein profiles of Gmm , Gpd , Gff and Gpg sialomes analyzed by SDS-PAGE analysis stained by Coomassie Blue. M indicates the Molecular Weight marker. Bands referred to in the sialome of each species are numbered from top to bottom. One representative image for the sialome data is shown. Additional results from SG extracts and replicate sialome samples are shown in S1 Fig .

    Journal: PLoS Neglected Tropical Diseases

    Article Title: Immunogenicity and Serological Cross-Reactivity of Saliva Proteins among Different Tsetse Species

    doi: 10.1371/journal.pntd.0004038

    Figure Lengend Snippet: SDS PAGE analysis of saliva from different tsetse species. Lanes 1–4 show protein profiles of Gmm , Gpd , Gff and Gpg sialomes analyzed by SDS-PAGE analysis stained by Coomassie Blue. M indicates the Molecular Weight marker. Bands referred to in the sialome of each species are numbered from top to bottom. One representative image for the sialome data is shown. Additional results from SG extracts and replicate sialome samples are shown in S1 Fig .

    Article Snippet: SDS Polyacrylamide Gel Electrophoresis (PAGE) analysis and immunoblotting Same amount of total sialome proteins obtained from dissected SG (or extracts from the same number of dissected salivary glands) were analyzed by 12% SDS-PAGE under reducing conditions and either stained by coomasie blue, or transferred to nitrocellulose membranes (BioRad, Cat # 162–0112) according to standard protocols [ ].

    Techniques: SDS Page, Staining, Molecular Weight, Marker

    ARF-GAPs mediate Arf1 Δ N17p-Q71L and coatomer binding to the cytosolic domains of v-SNAREs. SDS-PAGE analysis of in vitro binding of coatomer (top) and Arf1ΔN17p-Q71L (bottom) to GST (lanes 1 and 2) or GST fusions to v-SNAREs (lanes 3 and 4, Bet1p-GST; lanes 5 and 6, Sec22p-GST; lanes 7 and 8, GST-Bos1p). GST or GST fusion proteins were immobilized onto glutathione-agarose. Where indicated, 20 nM Glo3p (A) or Gcs1p (B) were added to the reaction, and 50% of the amount added is shown in lane 9. The guanine nucleotide on Arf1ΔN17p-Q71L was exchanged to GTP before the binding reaction, and 1.2 μM preexchanged Arf1ΔN17p-Q71L was added to the binding reaction. All reactions contained GTP. The coatomer concentration was 40 nM in the assay. After the binding reaction, the unbound proteins were removed by centrifugation. The proteins immobilized on the glutathione-agarose were separated by SDS-PAGE followed by SyproRed staining and analysis using the red fluorescent mode of a Storm PhosphorImager. In A, lanes 10 and 11 represent 20% of Arf1ΔN17p-Q71L and 50% of coatomer present in the reaction, respectively. In Bet1p-GST and Sec22p-GST, GST is the COOH-terminal fusion partner, whereas in GST-Bos1p it is in the NH 2 -terminal position.

    Journal: The Journal of Cell Biology

    Article Title: ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat

    doi: 10.1083/jcb.200112092

    Figure Lengend Snippet: ARF-GAPs mediate Arf1 Δ N17p-Q71L and coatomer binding to the cytosolic domains of v-SNAREs. SDS-PAGE analysis of in vitro binding of coatomer (top) and Arf1ΔN17p-Q71L (bottom) to GST (lanes 1 and 2) or GST fusions to v-SNAREs (lanes 3 and 4, Bet1p-GST; lanes 5 and 6, Sec22p-GST; lanes 7 and 8, GST-Bos1p). GST or GST fusion proteins were immobilized onto glutathione-agarose. Where indicated, 20 nM Glo3p (A) or Gcs1p (B) were added to the reaction, and 50% of the amount added is shown in lane 9. The guanine nucleotide on Arf1ΔN17p-Q71L was exchanged to GTP before the binding reaction, and 1.2 μM preexchanged Arf1ΔN17p-Q71L was added to the binding reaction. All reactions contained GTP. The coatomer concentration was 40 nM in the assay. After the binding reaction, the unbound proteins were removed by centrifugation. The proteins immobilized on the glutathione-agarose were separated by SDS-PAGE followed by SyproRed staining and analysis using the red fluorescent mode of a Storm PhosphorImager. In A, lanes 10 and 11 represent 20% of Arf1ΔN17p-Q71L and 50% of coatomer present in the reaction, respectively. In Bet1p-GST and Sec22p-GST, GST is the COOH-terminal fusion partner, whereas in GST-Bos1p it is in the NH 2 -terminal position.

    Article Snippet: Eluted proteins were analyzed by SDS gel electrophoresis, SyproRed staining, and scanning with a Storm PhosphorImager (Amersham Pharmacia Biotech).

    Techniques: Binding Assay, SDS Page, In Vitro, Concentration Assay, Centrifugation, Staining

    Glo3p induce conformational changes on v-SNARE–GST fusion proteins. (A) Bet1p-GST and Sec22p-GST were mock treated (−Glo3p) or pretreated with ARF-GAP (+Glo3p). The ARF-GAP was removed before the addition of the proteases to the SNAREs. Samples were withdrawn at the indicated time points and analyzed by SDS-PAGE and SyproRed staining. B shows a quantification of A. The standard derivation was calculated from six and nine experiments for Bet1p-GST and Sec22p-GST, respectively. (C) Sec23/24p complex can bind to Bet1p-(1–65)–GST even in the absence of Sar1p upon activation by Glo3p. Binding reactions were performed under the same conditions as above. Lane 5 shows 20% of the Sar1p that was present in the binding reactions. Sec23/24p complex was added to all reactions. (D) Binding of Sar1p and Sec23/24p complex is enhanced in the presence of Glo3p. Binding assays to Bet1p-GST were performed under optimized conditions for COPII binding as described by Springer and Schekman (1998) . GTP-γ–S was present in all reactions in C and D.

    Journal: The Journal of Cell Biology

    Article Title: ARF-GAP-mediated interaction between the ER-Golgi v-SNAREs and the COPI coat

    doi: 10.1083/jcb.200112092

    Figure Lengend Snippet: Glo3p induce conformational changes on v-SNARE–GST fusion proteins. (A) Bet1p-GST and Sec22p-GST were mock treated (−Glo3p) or pretreated with ARF-GAP (+Glo3p). The ARF-GAP was removed before the addition of the proteases to the SNAREs. Samples were withdrawn at the indicated time points and analyzed by SDS-PAGE and SyproRed staining. B shows a quantification of A. The standard derivation was calculated from six and nine experiments for Bet1p-GST and Sec22p-GST, respectively. (C) Sec23/24p complex can bind to Bet1p-(1–65)–GST even in the absence of Sar1p upon activation by Glo3p. Binding reactions were performed under the same conditions as above. Lane 5 shows 20% of the Sar1p that was present in the binding reactions. Sec23/24p complex was added to all reactions. (D) Binding of Sar1p and Sec23/24p complex is enhanced in the presence of Glo3p. Binding assays to Bet1p-GST were performed under optimized conditions for COPII binding as described by Springer and Schekman (1998) . GTP-γ–S was present in all reactions in C and D.

    Article Snippet: Eluted proteins were analyzed by SDS gel electrophoresis, SyproRed staining, and scanning with a Storm PhosphorImager (Amersham Pharmacia Biotech).

    Techniques: SDS Page, Staining, Activation Assay, Binding Assay

    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

    Envelope glycoprotein incorporation into virions. Virions were pelleted, lysed, separated by SDS-PAGE, and visualized by Western blotting. (A) Virion-associated gp120 was detected using anti-gp120 MAb 3.11H, and p27 capsid protein was detected by MAb 2F12. (B) Densitometric analysis [(variant gp120/p27)/(parental gp120/p27)] of bands in panel A.

    Journal: Journal of Virology

    Article Title: Infectivity and Neutralization of Simian Immunodeficiency Virus with FLAG Epitope Insertion in gp120 Variable Loops ▿

    doi: 10.1128/JVI.00831-07

    Figure Lengend Snippet: Envelope glycoprotein incorporation into virions. Virions were pelleted, lysed, separated by SDS-PAGE, and visualized by Western blotting. (A) Virion-associated gp120 was detected using anti-gp120 MAb 3.11H, and p27 capsid protein was detected by MAb 2F12. (B) Densitometric analysis [(variant gp120/p27)/(parental gp120/p27)] of bands in panel A.

    Article Snippet: The samples were resolved in an 8 to 16% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gradient gel (Invitrogen, Carlsbad, CA), transferred, and blocked as described above.

    Techniques: SDS Page, Western Blot, Variant Assay

    The effect of o -methylated 3HPs on: i) the HEWL fibril formation, ii) its α-to-β conformational rearrangement, iii) the HEWL acid-induced proteolysis, and iv) the HEWL thermal stability. (A–B) AFM micrographs of HEWL solutions (0.2 mM) previously incubated during 16d at pH 2.0 and 60 °C either (A) in buffer, or (B) in the presence of 20 mM 2 m-3HP. The scale bar represents 0.5 μm. (C-D) Far-UV CD spectra of HEWL incubated during 0, 7 and 17 days at pH 2.0 and 60 °C either (C) in buffer, or (D) in the presence of 20 mM 2 m-3HP. (E) SDS-PAGE gel analysis of solutions containing HEWL incubated at pH 2.0 and 60 °C during 0 and 10d either in the absence or in the presence of different o -methylated 3HPs. (F) Differential scanning calorimetry thermograms of different HEWL solutions (0.2 mM) at pH 2.0 acquired in the absence or in the presence of 20 mM of the different o -methylated 3HPs.

    Journal: Scientific Reports

    Article Title: Ortho-methylated 3-hydroxypyridines hinder hen egg-white lysozyme fibrillogenesis

    doi: 10.1038/srep12052

    Figure Lengend Snippet: The effect of o -methylated 3HPs on: i) the HEWL fibril formation, ii) its α-to-β conformational rearrangement, iii) the HEWL acid-induced proteolysis, and iv) the HEWL thermal stability. (A–B) AFM micrographs of HEWL solutions (0.2 mM) previously incubated during 16d at pH 2.0 and 60 °C either (A) in buffer, or (B) in the presence of 20 mM 2 m-3HP. The scale bar represents 0.5 μm. (C-D) Far-UV CD spectra of HEWL incubated during 0, 7 and 17 days at pH 2.0 and 60 °C either (C) in buffer, or (D) in the presence of 20 mM 2 m-3HP. (E) SDS-PAGE gel analysis of solutions containing HEWL incubated at pH 2.0 and 60 °C during 0 and 10d either in the absence or in the presence of different o -methylated 3HPs. (F) Differential scanning calorimetry thermograms of different HEWL solutions (0.2 mM) at pH 2.0 acquired in the absence or in the presence of 20 mM of the different o -methylated 3HPs.

    Article Snippet: SDS-PAGE electrophoresis Aliquots (4 μl) corresponding to the different HEWL/HP reaction mixtures were taken at 0 and 10d of incubation and mixed with 4 μl of Laemmli sample buffer (Bio-Rad) containing 50mM DTT.

    Techniques: Methylation, Incubation, SDS Page