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Santa Cruz Biotechnology nonidet p 40
Localization of SUMO-1-modified HIPK2 to nuclear speckles (dots). ( A ) Fractionation of SUMO-1-modified HIPK2. Cellular extracts from CV-1 cells cotransfected with GFP-SUMO-1 and Myc-HIPK2 expression vectors were fractionated. Proteins from each fraction were separated on SDS/polyacrylamide gradient (4–12%) gels and analyzed by Western blotting using a mouse anti-Myc Ab. HIPK2 and SUMO-1-modified HIPK2 are detected in the nuclear fraction (lane 3) and in the Nonidet P-40-insoluble fractions (lanes 7 and 8). SUMO-1-modified HIPK2 and unmodified HIPK2 are marked by bracket and arrowhead, respectively. ( B ) Confocal microscopic image of the nucleus showing colocalization of HIPK2 and SUMO-1 to nuclear speckles (dots). CV-1 cells overexpressing Myc-HIPK2 and GFP-SUMO-1 were fixed and subjected to immunofluorescence staining with a mouse anti-Myc mAb. The red signal (HIPK2) is obtained with an anti-mouse IgG rhodamine red-conjugated secondary Ab, whereas the green signal (SUMO-1) is obtained with GFP fluorescence. Superimposing two colors (MERGE) results in a yellow signal, indicating colocalization of the two proteins.
Nonidet P 40, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Covalent modification of the homeodomain-interacting protein kinase 2 (HIPK2) by the ubiquitin-like protein SUMO-1"

Article Title: Covalent modification of the homeodomain-interacting protein kinase 2 (HIPK2) by the ubiquitin-like protein SUMO-1

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Localization of SUMO-1-modified HIPK2 to nuclear speckles (dots). ( A ) Fractionation of SUMO-1-modified HIPK2. Cellular extracts from CV-1 cells cotransfected with GFP-SUMO-1 and Myc-HIPK2 expression vectors were fractionated. Proteins from each fraction were separated on SDS/polyacrylamide gradient (4–12%) gels and analyzed by Western blotting using a mouse anti-Myc Ab. HIPK2 and SUMO-1-modified HIPK2 are detected in the nuclear fraction (lane 3) and in the Nonidet P-40-insoluble fractions (lanes 7 and 8). SUMO-1-modified HIPK2 and unmodified HIPK2 are marked by bracket and arrowhead, respectively. ( B ) Confocal microscopic image of the nucleus showing colocalization of HIPK2 and SUMO-1 to nuclear speckles (dots). CV-1 cells overexpressing Myc-HIPK2 and GFP-SUMO-1 were fixed and subjected to immunofluorescence staining with a mouse anti-Myc mAb. The red signal (HIPK2) is obtained with an anti-mouse IgG rhodamine red-conjugated secondary Ab, whereas the green signal (SUMO-1) is obtained with GFP fluorescence. Superimposing two colors (MERGE) results in a yellow signal, indicating colocalization of the two proteins.
Figure Legend Snippet: Localization of SUMO-1-modified HIPK2 to nuclear speckles (dots). ( A ) Fractionation of SUMO-1-modified HIPK2. Cellular extracts from CV-1 cells cotransfected with GFP-SUMO-1 and Myc-HIPK2 expression vectors were fractionated. Proteins from each fraction were separated on SDS/polyacrylamide gradient (4–12%) gels and analyzed by Western blotting using a mouse anti-Myc Ab. HIPK2 and SUMO-1-modified HIPK2 are detected in the nuclear fraction (lane 3) and in the Nonidet P-40-insoluble fractions (lanes 7 and 8). SUMO-1-modified HIPK2 and unmodified HIPK2 are marked by bracket and arrowhead, respectively. ( B ) Confocal microscopic image of the nucleus showing colocalization of HIPK2 and SUMO-1 to nuclear speckles (dots). CV-1 cells overexpressing Myc-HIPK2 and GFP-SUMO-1 were fixed and subjected to immunofluorescence staining with a mouse anti-Myc mAb. The red signal (HIPK2) is obtained with an anti-mouse IgG rhodamine red-conjugated secondary Ab, whereas the green signal (SUMO-1) is obtained with GFP fluorescence. Superimposing two colors (MERGE) results in a yellow signal, indicating colocalization of the two proteins.

Techniques Used: Modification, Fractionation, Expressing, Western Blot, Immunofluorescence, Staining, Fluorescence

2) Product Images from "A PGC-1α-O-GlcNAc Transferase Complex Regulates FoxO Transcription Factor Activity in Response to Glucose"

Article Title: A PGC-1α-O-GlcNAc Transferase Complex Regulates FoxO Transcription Factor Activity in Response to Glucose

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M808890200

PGC-1α interacts with OGT. A , Fao cells were infected  with Ad-FLAG-PGC-1α and treated with insulin for 1 h prior to harvesting  and OGT co-immunoprecipitation ( IP ). Immunoprecipitates were  subjected to SDS-PAGE and immunoblotted ( IB ) for the presence of  PGC-1α. Membranes were then stripped and blotted for OGT. Immunoblots  are representative of three experiments.  B , Fao cells were infected  with Ad-FLAG-PGC-1α and treated with insulin for 1 h prior to harvesting  and FLAG co-immunoprecipitation. Immunoprecipitates were subjected to SDS-PAGE  and immunoblotted for the presence of OGT. Membranes were then stripped and  blotted for PGC-1α.  C , gel filtration chromatography (SMART  system, Superdex 200 column, phosphate-buffered saline, 1% Nonidet P-40  buffer) of lysates from rat liver incubated on ice for 30 min with either  normal IgG or anti-OGT antibodies (AL28). Fractions were subjected to SDS-PAGE  and blotted using anti-PGC-1α, anti-OGT (DM-17), and anti CPB. Data are  representative of two experiments.
Figure Legend Snippet: PGC-1α interacts with OGT. A , Fao cells were infected with Ad-FLAG-PGC-1α and treated with insulin for 1 h prior to harvesting and OGT co-immunoprecipitation ( IP ). Immunoprecipitates were subjected to SDS-PAGE and immunoblotted ( IB ) for the presence of PGC-1α. Membranes were then stripped and blotted for OGT. Immunoblots are representative of three experiments. B , Fao cells were infected with Ad-FLAG-PGC-1α and treated with insulin for 1 h prior to harvesting and FLAG co-immunoprecipitation. Immunoprecipitates were subjected to SDS-PAGE and immunoblotted for the presence of OGT. Membranes were then stripped and blotted for PGC-1α. C , gel filtration chromatography (SMART system, Superdex 200 column, phosphate-buffered saline, 1% Nonidet P-40 buffer) of lysates from rat liver incubated on ice for 30 min with either normal IgG or anti-OGT antibodies (AL28). Fractions were subjected to SDS-PAGE and blotted using anti-PGC-1α, anti-OGT (DM-17), and anti CPB. Data are representative of two experiments.

Techniques Used: Pyrolysis Gas Chromatography, Infection, Immunoprecipitation, SDS Page, Western Blot, Filtration, Chromatography, Incubation

Related Articles

Centrifugation:

Article Title: DNA-PKcs PARylation regulates DNA-PK kinase activity in the DNA damage response
Article Snippet: .. Immunoprecipitation and western blotting NETN buffer 300 [20 mM Tris-HCL (pH 8.0), 300 mM NaCl, 1 mM EDTA and 0.5% Nonidet P-40] was used to lyse the cells at 4°C for 10 min. Then NETN buffer 100 [20 mM Tris-HCL (pH 8.0), 100 mM NaCl, 1 mM EDTA and 0.5% Nonidet P-40] was used to lyse the cells at 4°C for 5 min. After the removal of the cell debris by centrifugation (12,000 × g for 10 min at 4°C), the supernatant was collected and incubated with IgG (1 µg/ml) and protein A/G (Santa Cruz Biotechnology, Inc.; 20 µl) with rotation for 1 h at 4°C for preclearing. .. Then, the precipitate was removed by centrifugation (12,000 × g for 10 min at 4°C) and the supernatant was collected and incubated with an antibody against DNA-PKcs (1 µg/ml) and protein A/G (Santa Cruz Biotechnology, Inc.; 40 µl) with rotation overnight at 4°C.

Western Blot:

Article Title: DNA-PKcs PARylation regulates DNA-PK kinase activity in the DNA damage response
Article Snippet: .. Immunoprecipitation and western blotting NETN buffer 300 [20 mM Tris-HCL (pH 8.0), 300 mM NaCl, 1 mM EDTA and 0.5% Nonidet P-40] was used to lyse the cells at 4°C for 10 min. Then NETN buffer 100 [20 mM Tris-HCL (pH 8.0), 100 mM NaCl, 1 mM EDTA and 0.5% Nonidet P-40] was used to lyse the cells at 4°C for 5 min. After the removal of the cell debris by centrifugation (12,000 × g for 10 min at 4°C), the supernatant was collected and incubated with IgG (1 µg/ml) and protein A/G (Santa Cruz Biotechnology, Inc.; 20 µl) with rotation for 1 h at 4°C for preclearing. .. Then, the precipitate was removed by centrifugation (12,000 × g for 10 min at 4°C) and the supernatant was collected and incubated with an antibody against DNA-PKcs (1 µg/ml) and protein A/G (Santa Cruz Biotechnology, Inc.; 40 µl) with rotation overnight at 4°C.

Transfection:

Article Title: Wnt-induced Vangl2 phosphorylation is dose-dependently required for planar cell polarity in mammalian development
Article Snippet: .. In the Co-IP experiment, transfected HEK293T cells were lysed in lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% Nonidet P-40) with protease and phosphatase inhibitors and pre-cleared by Protein A/G agarose (Santa Cruz). .. The cell lysates were then incubated with the anti-HA antibody (Roche, 0.5 μg) overnight at 4 °C followed by 2 h with Protein A/G agarose (Santa Cruz).

Immunoprecipitation:

Article Title: Covalent modification of the homeodomain-interacting protein kinase 2 (HIPK2) by the ubiquitin-like protein SUMO-1
Article Snippet: .. For immunoprecipitation, cell extracts (500 μg) were diluted (1:10) in 1× NETN [50 mM Tris⋅HCl (pH 7.5)/5 mM EDTA/300 mM NaCl/1 mM DTT/1% Nonidet P-40] containing protease inhibitors and incubated with an anti-GFP rabbit polyclonal Ab (10 μg/ml) or anti-SUMO-1 goat Ab (Santa Cruz Biotechnology) for 1 hr at 4°C. .. After adding protein A/G plus agarose beads (Santa Cruz Biotechnology), the mixtures were further incubated for 4 hr at 4°C with mild agitation.

Article Title: DNA-PKcs PARylation regulates DNA-PK kinase activity in the DNA damage response
Article Snippet: .. Immunoprecipitation and western blotting NETN buffer 300 [20 mM Tris-HCL (pH 8.0), 300 mM NaCl, 1 mM EDTA and 0.5% Nonidet P-40] was used to lyse the cells at 4°C for 10 min. Then NETN buffer 100 [20 mM Tris-HCL (pH 8.0), 100 mM NaCl, 1 mM EDTA and 0.5% Nonidet P-40] was used to lyse the cells at 4°C for 5 min. After the removal of the cell debris by centrifugation (12,000 × g for 10 min at 4°C), the supernatant was collected and incubated with IgG (1 µg/ml) and protein A/G (Santa Cruz Biotechnology, Inc.; 20 µl) with rotation for 1 h at 4°C for preclearing. .. Then, the precipitate was removed by centrifugation (12,000 × g for 10 min at 4°C) and the supernatant was collected and incubated with an antibody against DNA-PKcs (1 µg/ml) and protein A/G (Santa Cruz Biotechnology, Inc.; 40 µl) with rotation overnight at 4°C.

Incubation:

Article Title: A PGC-1α-O-GlcNAc Transferase Complex Regulates FoxO Transcription Factor Activity in Response to Glucose
Article Snippet: .. Rat liver was lysed in Tris-buffered saline with 1% Nonidet P-40 and incubated for 1 h on ice with either normal rabit IgG (Santa Cruz Biotechnology) or anti-Ogt (AL28) ( ). .. OGT Assays —The plasmid expressing recombinant OGT was a kind gift of Suzanne Walker ( ).

Article Title: Covalent modification of the homeodomain-interacting protein kinase 2 (HIPK2) by the ubiquitin-like protein SUMO-1
Article Snippet: .. For immunoprecipitation, cell extracts (500 μg) were diluted (1:10) in 1× NETN [50 mM Tris⋅HCl (pH 7.5)/5 mM EDTA/300 mM NaCl/1 mM DTT/1% Nonidet P-40] containing protease inhibitors and incubated with an anti-GFP rabbit polyclonal Ab (10 μg/ml) or anti-SUMO-1 goat Ab (Santa Cruz Biotechnology) for 1 hr at 4°C. .. After adding protein A/G plus agarose beads (Santa Cruz Biotechnology), the mixtures were further incubated for 4 hr at 4°C with mild agitation.

Article Title: Nuclear Export-independent Inhibition of Foxa2 by Insulin *
Article Snippet: .. 9 μ m cryosections were fixed in 4% paraformaldehyde in PBS at 4 °C for 30 min, permeabilized in 0.2% Nonidet P-40 in PBS, blocked in 5% normal donkey serum, 1% bovine serum albumin, 0.1% Nonidet P-40 in PBS, and incubated with anti-HA antibody (1:25, Santa Cruz Biotechnology, sc-805) overnight at 4 °C in a humidified chamber. .. Donkey anti-rabbit IgG Alexa Fluor 488 (Molecular Probes) was used as a secondary antibody and mounted with VECTASHIELD mounting media with 4,6-diamidino-2-phenylindole (Vector Laboratories) to visualize nuclei.

Article Title: Translational Homeostasis: Eukaryotic Translation Initiation Factor 4E Control of 4E-Binding Protein 1 and p70 S6 Kinase Activities
Article Snippet: .. Cell extract (20 μg) was diluted with extraction buffer (50 mM Tris-HCl, pH 8.0; 120 mM NaCl; 20 mM NaF; 1 mM benzamidine; 1 mM EDTA; 6 mM EGTA, 1% [vol/vol] Nonidet P-40; 0.1 mM PMSF) and incubated with rabbit polyclonal anti-p70S6k antibody (Santa Cruz Biotechnology) on ice for 2 h. A 37.5% suspension of protein A-Sepharose (Repligen) resin was added and incubated end over end at 4°C for 1 h. The resin was washed twice with extraction buffer and once with dilution buffer (50 mM morpholinepropanesulfonic acid [MOPS], pH 7.2; 5 mM MgCl2 ; 10 mM NaF; 30 mM β-glycerophosphate). p70S6k activity was assayed with 40S ribosomal subunits in a mixture (10 μl) containing 50 mM MOPS, pH 7.2; 1 mM DTT; 5 mM MgCl2 ; 5 mM p -nitrophenyl phosphate; 100 μM ATP; 0.5 μM protein kinase inhibitor (Sigma); 6 μCi of [32 P]-ATP; 20 μg of 40S ribosomal subunits ( ). ..

Article Title: The Role of PTP1B O-GlcNAcylation in Hepatic Insulin Resistance
Article Snippet: .. After 36 h, cells were collected in 50 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40 with protease inhibitors and then lysed by vortexing for 2 h. Equal amounts of lysate were incubated overnight with 5 μL of anti-O -GlcNAc antibodies rotating at 4 °C, followed by incubation with 30 μL of protein A/G PLUS-agarose (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 3 h at 4 °C. ..

Article Title: DNA-PKcs PARylation regulates DNA-PK kinase activity in the DNA damage response
Article Snippet: .. Immunoprecipitation and western blotting NETN buffer 300 [20 mM Tris-HCL (pH 8.0), 300 mM NaCl, 1 mM EDTA and 0.5% Nonidet P-40] was used to lyse the cells at 4°C for 10 min. Then NETN buffer 100 [20 mM Tris-HCL (pH 8.0), 100 mM NaCl, 1 mM EDTA and 0.5% Nonidet P-40] was used to lyse the cells at 4°C for 5 min. After the removal of the cell debris by centrifugation (12,000 × g for 10 min at 4°C), the supernatant was collected and incubated with IgG (1 µg/ml) and protein A/G (Santa Cruz Biotechnology, Inc.; 20 µl) with rotation for 1 h at 4°C for preclearing. .. Then, the precipitate was removed by centrifugation (12,000 × g for 10 min at 4°C) and the supernatant was collected and incubated with an antibody against DNA-PKcs (1 µg/ml) and protein A/G (Santa Cruz Biotechnology, Inc.; 40 µl) with rotation overnight at 4°C.

Activity Assay:

Article Title: Translational Homeostasis: Eukaryotic Translation Initiation Factor 4E Control of 4E-Binding Protein 1 and p70 S6 Kinase Activities
Article Snippet: .. Cell extract (20 μg) was diluted with extraction buffer (50 mM Tris-HCl, pH 8.0; 120 mM NaCl; 20 mM NaF; 1 mM benzamidine; 1 mM EDTA; 6 mM EGTA, 1% [vol/vol] Nonidet P-40; 0.1 mM PMSF) and incubated with rabbit polyclonal anti-p70S6k antibody (Santa Cruz Biotechnology) on ice for 2 h. A 37.5% suspension of protein A-Sepharose (Repligen) resin was added and incubated end over end at 4°C for 1 h. The resin was washed twice with extraction buffer and once with dilution buffer (50 mM morpholinepropanesulfonic acid [MOPS], pH 7.2; 5 mM MgCl2 ; 10 mM NaF; 30 mM β-glycerophosphate). p70S6k activity was assayed with 40S ribosomal subunits in a mixture (10 μl) containing 50 mM MOPS, pH 7.2; 1 mM DTT; 5 mM MgCl2 ; 5 mM p -nitrophenyl phosphate; 100 μM ATP; 0.5 μM protein kinase inhibitor (Sigma); 6 μCi of [32 P]-ATP; 20 μg of 40S ribosomal subunits ( ). ..

Co-Immunoprecipitation Assay:

Article Title: Wnt-induced Vangl2 phosphorylation is dose-dependently required for planar cell polarity in mammalian development
Article Snippet: .. In the Co-IP experiment, transfected HEK293T cells were lysed in lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% Nonidet P-40) with protease and phosphatase inhibitors and pre-cleared by Protein A/G agarose (Santa Cruz). .. The cell lysates were then incubated with the anti-HA antibody (Roche, 0.5 μg) overnight at 4 °C followed by 2 h with Protein A/G agarose (Santa Cruz).

Lysis:

Article Title: Wnt-induced Vangl2 phosphorylation is dose-dependently required for planar cell polarity in mammalian development
Article Snippet: .. In the Co-IP experiment, transfected HEK293T cells were lysed in lysis buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% Nonidet P-40) with protease and phosphatase inhibitors and pre-cleared by Protein A/G agarose (Santa Cruz). .. The cell lysates were then incubated with the anti-HA antibody (Roche, 0.5 μg) overnight at 4 °C followed by 2 h with Protein A/G agarose (Santa Cruz).

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    Santa Cruz Biotechnology np 40 soluble fractions
    16E1 ∧ E4 decreases the solubility of active Cdk1/cyclin B1. (A) G 1 /S-synchronized SiHa cells expressing Myt1, 16E1 ∧ E4, or β-galactosidase were harvested at 20 h post-block release and fractionated with 1% <t>NP-40</t> (NP-40-soluble fraction)
    Np 40 Soluble Fractions, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology np 40 lysates
    Analysis of eIF4E interaction with 4E-BP1 and eIF4G during hypoxia. (A) Equal amounts of <t>NP-40</t> lysates (300 μg) from MCF10A, CRL2324, and HTB20 cells under normoxic (N) or hypoxic (H) conditions were subjected to m 7 GTP-Sepharose cap-chromatography,
    Np 40 Lysates, supplied by Santa Cruz Biotechnology, 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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    Santa Cruz Biotechnology nonidet p 40
    Localization of SUMO-1-modified HIPK2 to nuclear speckles (dots). ( A ) Fractionation of SUMO-1-modified HIPK2. Cellular extracts from CV-1 cells cotransfected with GFP-SUMO-1 and Myc-HIPK2 expression vectors were fractionated. Proteins from each fraction were separated on SDS/polyacrylamide gradient (4–12%) gels and analyzed by Western blotting using a mouse anti-Myc Ab. HIPK2 and SUMO-1-modified HIPK2 are detected in the nuclear fraction (lane 3) and in the Nonidet P-40-insoluble fractions (lanes 7 and 8). SUMO-1-modified HIPK2 and unmodified HIPK2 are marked by bracket and arrowhead, respectively. ( B ) Confocal microscopic image of the nucleus showing colocalization of HIPK2 and SUMO-1 to nuclear speckles (dots). CV-1 cells overexpressing Myc-HIPK2 and GFP-SUMO-1 were fixed and subjected to immunofluorescence staining with a mouse anti-Myc mAb. The red signal (HIPK2) is obtained with an anti-mouse IgG rhodamine red-conjugated secondary Ab, whereas the green signal (SUMO-1) is obtained with GFP fluorescence. Superimposing two colors (MERGE) results in a yellow signal, indicating colocalization of the two proteins.
    Nonidet P 40, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology np 40 buffer
    Levels of activated Fgfrs and activated EphA2 are altered in Dlg f/f 10Cre lenses. ( A ) Lenses from P10 control and Dlg f/f 10Cre lenses were extracted with Triton X-100, and the pellets were resuspended in <t>NP-40</t> buffer. The extracts were subjected to immunoprecipitation (IP) with antibodies against the indicated proteins followed by Western blotting with antibody against phosphotyrosine (IB: p-Tyr). As a loading control, blots were reprobed for their respective immunoprecipitated receptor proteins. ( B ) Quantification of protein levels. Shown are the levels of the indicated proteins in extracts from Dlg f/f 10Cre lenses relative to levels in the controls (control levels set a 1.0). Signal intensities were quantified by phosphorimager analysis, and the data were subjected to statistical analysis as described in Materials and Methods. At least three protein pools were immunoprecipitated and immunoblotted in triplicate over one to three blots. Error bars denote SD. *FDR
    Np 40 Buffer, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    16E1 ∧ E4 decreases the solubility of active Cdk1/cyclin B1. (A) G 1 /S-synchronized SiHa cells expressing Myt1, 16E1 ∧ E4, or β-galactosidase were harvested at 20 h post-block release and fractionated with 1% NP-40 (NP-40-soluble fraction)

    Journal:

    Article Title: Human Papillomavirus Type 16 E1∧E4-Induced G2 Arrest Is Associated with Cytoplasmic Retention of Active Cdk1/Cyclin B1 Complexes

    doi: 10.1128/JVI.79.7.3998-4011.2005

    Figure Lengend Snippet: 16E1 ∧ E4 decreases the solubility of active Cdk1/cyclin B1. (A) G 1 /S-synchronized SiHa cells expressing Myt1, 16E1 ∧ E4, or β-galactosidase were harvested at 20 h post-block release and fractionated with 1% NP-40 (NP-40-soluble fraction)

    Article Snippet: To coimmunoprecipitate 16E1∧ E4 and cyclin B1, 1% NP-40-soluble fractions containing 0.25% gelatin were rotated with 10 μl of anti-cyclin B1 polyclonal antibody clone H-433 (Santa Cruz) or normal rabbit serum (Sigma) for 1 h at 4°C.

    Techniques: Solubility, Expressing, Blocking Assay

    Analysis of eIF4E interaction with 4E-BP1 and eIF4G during hypoxia. (A) Equal amounts of NP-40 lysates (300 μg) from MCF10A, CRL2324, and HTB20 cells under normoxic (N) or hypoxic (H) conditions were subjected to m 7 GTP-Sepharose cap-chromatography,

    Journal:

    Article Title: Hypoxia Inhibits Protein Synthesis through a 4E-BP1 and Elongation Factor 2 Kinase Pathway Controlled by mTOR and Uncoupled in Breast Cancer Cells †

    doi: 10.1128/MCB.26.10.3955-3965.2006

    Figure Lengend Snippet: Analysis of eIF4E interaction with 4E-BP1 and eIF4G during hypoxia. (A) Equal amounts of NP-40 lysates (300 μg) from MCF10A, CRL2324, and HTB20 cells under normoxic (N) or hypoxic (H) conditions were subjected to m 7 GTP-Sepharose cap-chromatography,

    Article Snippet: Equal amounts of protein from NP-40 lysates were precleared for 1 h at 4°C with 30 μl of protein A-Sepharose (Santa Cruz Biotech) and then incubated overnight with the indicated antiserum (preimmune serum or anti-eIF4G C-terminal fragment) at 4°C.

    Techniques: Chromatography

    Localization of SUMO-1-modified HIPK2 to nuclear speckles (dots). ( A ) Fractionation of SUMO-1-modified HIPK2. Cellular extracts from CV-1 cells cotransfected with GFP-SUMO-1 and Myc-HIPK2 expression vectors were fractionated. Proteins from each fraction were separated on SDS/polyacrylamide gradient (4–12%) gels and analyzed by Western blotting using a mouse anti-Myc Ab. HIPK2 and SUMO-1-modified HIPK2 are detected in the nuclear fraction (lane 3) and in the Nonidet P-40-insoluble fractions (lanes 7 and 8). SUMO-1-modified HIPK2 and unmodified HIPK2 are marked by bracket and arrowhead, respectively. ( B ) Confocal microscopic image of the nucleus showing colocalization of HIPK2 and SUMO-1 to nuclear speckles (dots). CV-1 cells overexpressing Myc-HIPK2 and GFP-SUMO-1 were fixed and subjected to immunofluorescence staining with a mouse anti-Myc mAb. The red signal (HIPK2) is obtained with an anti-mouse IgG rhodamine red-conjugated secondary Ab, whereas the green signal (SUMO-1) is obtained with GFP fluorescence. Superimposing two colors (MERGE) results in a yellow signal, indicating colocalization of the two proteins.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Covalent modification of the homeodomain-interacting protein kinase 2 (HIPK2) by the ubiquitin-like protein SUMO-1

    doi:

    Figure Lengend Snippet: Localization of SUMO-1-modified HIPK2 to nuclear speckles (dots). ( A ) Fractionation of SUMO-1-modified HIPK2. Cellular extracts from CV-1 cells cotransfected with GFP-SUMO-1 and Myc-HIPK2 expression vectors were fractionated. Proteins from each fraction were separated on SDS/polyacrylamide gradient (4–12%) gels and analyzed by Western blotting using a mouse anti-Myc Ab. HIPK2 and SUMO-1-modified HIPK2 are detected in the nuclear fraction (lane 3) and in the Nonidet P-40-insoluble fractions (lanes 7 and 8). SUMO-1-modified HIPK2 and unmodified HIPK2 are marked by bracket and arrowhead, respectively. ( B ) Confocal microscopic image of the nucleus showing colocalization of HIPK2 and SUMO-1 to nuclear speckles (dots). CV-1 cells overexpressing Myc-HIPK2 and GFP-SUMO-1 were fixed and subjected to immunofluorescence staining with a mouse anti-Myc mAb. The red signal (HIPK2) is obtained with an anti-mouse IgG rhodamine red-conjugated secondary Ab, whereas the green signal (SUMO-1) is obtained with GFP fluorescence. Superimposing two colors (MERGE) results in a yellow signal, indicating colocalization of the two proteins.

    Article Snippet: For immunoprecipitation, cell extracts (500 μg) were diluted (1:10) in 1× NETN [50 mM Tris⋅HCl (pH 7.5)/5 mM EDTA/300 mM NaCl/1 mM DTT/1% Nonidet P-40] containing protease inhibitors and incubated with an anti-GFP rabbit polyclonal Ab (10 μg/ml) or anti-SUMO-1 goat Ab (Santa Cruz Biotechnology) for 1 hr at 4°C.

    Techniques: Modification, Fractionation, Expressing, Western Blot, Immunofluorescence, Staining, Fluorescence

    Levels of activated Fgfrs and activated EphA2 are altered in Dlg f/f 10Cre lenses. ( A ) Lenses from P10 control and Dlg f/f 10Cre lenses were extracted with Triton X-100, and the pellets were resuspended in NP-40 buffer. The extracts were subjected to immunoprecipitation (IP) with antibodies against the indicated proteins followed by Western blotting with antibody against phosphotyrosine (IB: p-Tyr). As a loading control, blots were reprobed for their respective immunoprecipitated receptor proteins. ( B ) Quantification of protein levels. Shown are the levels of the indicated proteins in extracts from Dlg f/f 10Cre lenses relative to levels in the controls (control levels set a 1.0). Signal intensities were quantified by phosphorimager analysis, and the data were subjected to statistical analysis as described in Materials and Methods. At least three protein pools were immunoprecipitated and immunoblotted in triplicate over one to three blots. Error bars denote SD. *FDR

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Dlg-1 Interacts With and Regulates the Activities of Fibroblast Growth Factor Receptors and EphA2 in the Mouse Lens

    doi: 10.1167/iovs.15-17727

    Figure Lengend Snippet: Levels of activated Fgfrs and activated EphA2 are altered in Dlg f/f 10Cre lenses. ( A ) Lenses from P10 control and Dlg f/f 10Cre lenses were extracted with Triton X-100, and the pellets were resuspended in NP-40 buffer. The extracts were subjected to immunoprecipitation (IP) with antibodies against the indicated proteins followed by Western blotting with antibody against phosphotyrosine (IB: p-Tyr). As a loading control, blots were reprobed for their respective immunoprecipitated receptor proteins. ( B ) Quantification of protein levels. Shown are the levels of the indicated proteins in extracts from Dlg f/f 10Cre lenses relative to levels in the controls (control levels set a 1.0). Signal intensities were quantified by phosphorimager analysis, and the data were subjected to statistical analysis as described in Materials and Methods. At least three protein pools were immunoprecipitated and immunoblotted in triplicate over one to three blots. Error bars denote SD. *FDR

    Article Snippet: Protein A Sepharose or Protein G Sepharose pellets were washed with NP-40 buffer and resuspended in urea buffer containing loading dye, and proteins were denatured at 95°C, fractionated on 7.5% SDS/PAGE, transferred to PVDF membranes, and immunoblotted for β-catenin, Dlg-1, Fgfr1, Fgfr2, Fgfr3, EphA2, and mouse anti-pTyr (Santa Cruz Biotechnology Cat# SC-508) antibody as described above.

    Techniques: Immunoprecipitation, Western Blot

    Levels of protein–protein interactions are altered in Dlg f/f 10Cre lenses. ( A ) Lenses from P10 control and Dlg10 f/f Cre mice were extracted with Triton X-100, and the pellets were resuspended in NP-40 buffer. Extracts were subjected to immunoprecipitation and immunoblot using antibodies against the indicated proteins. As a loading control, the blots were reprobed for the respective immunoprecipitated proteins. ( B ) Quantification of protein levels. Shown are the levels of the indicated coimmunoprecipitated proteins in Dlg10 f/f Cre extracts compared with control (normalized to the levels of reprobed protein in Dlg10 f/f Cre extracts compared with control). Signal intensities were quantified by phosphorimager analysis, and the data were subjected to statistical analysis as described in Materials and Methods. In control lenses, β-catenin interacts with N-cadherin, Dlg-1 interacts with β-catenin, EphA2, Fgfr1, Fgfr2, and Fgfr3, and EphA2 interacts with β-catenin, Fgfr1, Fgfr2, and Fgfr3. In Dlg f/f 10Cre lenses, the interactions between β-catenin and N-cadherin, β-catenin and EphA2, N-cadherin and EphA2, and EphA2 and Fgfr2 were reduced compared with controls, whereas the interaction between EphA2 and Fgfr1 and EphA2 and Fgfr3 were increased compared with controls. Error bars denote SD. *FDR

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Dlg-1 Interacts With and Regulates the Activities of Fibroblast Growth Factor Receptors and EphA2 in the Mouse Lens

    doi: 10.1167/iovs.15-17727

    Figure Lengend Snippet: Levels of protein–protein interactions are altered in Dlg f/f 10Cre lenses. ( A ) Lenses from P10 control and Dlg10 f/f Cre mice were extracted with Triton X-100, and the pellets were resuspended in NP-40 buffer. Extracts were subjected to immunoprecipitation and immunoblot using antibodies against the indicated proteins. As a loading control, the blots were reprobed for the respective immunoprecipitated proteins. ( B ) Quantification of protein levels. Shown are the levels of the indicated coimmunoprecipitated proteins in Dlg10 f/f Cre extracts compared with control (normalized to the levels of reprobed protein in Dlg10 f/f Cre extracts compared with control). Signal intensities were quantified by phosphorimager analysis, and the data were subjected to statistical analysis as described in Materials and Methods. In control lenses, β-catenin interacts with N-cadherin, Dlg-1 interacts with β-catenin, EphA2, Fgfr1, Fgfr2, and Fgfr3, and EphA2 interacts with β-catenin, Fgfr1, Fgfr2, and Fgfr3. In Dlg f/f 10Cre lenses, the interactions between β-catenin and N-cadherin, β-catenin and EphA2, N-cadherin and EphA2, and EphA2 and Fgfr2 were reduced compared with controls, whereas the interaction between EphA2 and Fgfr1 and EphA2 and Fgfr3 were increased compared with controls. Error bars denote SD. *FDR

    Article Snippet: Protein A Sepharose or Protein G Sepharose pellets were washed with NP-40 buffer and resuspended in urea buffer containing loading dye, and proteins were denatured at 95°C, fractionated on 7.5% SDS/PAGE, transferred to PVDF membranes, and immunoblotted for β-catenin, Dlg-1, Fgfr1, Fgfr2, Fgfr3, EphA2, and mouse anti-pTyr (Santa Cruz Biotechnology Cat# SC-508) antibody as described above.

    Techniques: Mouse Assay, Immunoprecipitation