nonidet p40  (Millipore)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    Name:
    Nonidet P 40
    Description:

    Catalog Number:
    21-3277
    Price:
    None
    Buy from Supplier


    Structured Review

    Millipore nonidet p40
    Nonidet P 40

    https://www.bioz.com/result/nonidet p40/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nonidet p40 - by Bioz Stars, 2021-05
    97/100 stars

    Images

    Related Articles

    Immunoprecipitation:

    Article Title: O-GlcNAcylation of SIRT1 enhances its deacetylase activity and promotes cytoprotection under stress
    Article Snippet: Stable cell lines were obtained by retroviral or lentiviral infection and selection with puromycin (1 μg ml−1 ) or hygromycin B (200 μg ml−1 ) for 2 weeks. .. Immunoprecipitation (IP) and CoIP For IP assay, cells were lysed in RIPA buffer (25 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP40, 0.1% SDS, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF, 10 μM PUGNAc [Sigma], 2 mM STZ [Sigma], 10 μM TMG [Sigma], 40 mM GlcNAc [Sigma], and Complete™ protease inhibitors [Roche]). .. For CoIP assay, cells were lysed in NP40 lysis buffer (25 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP40, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF, 10 μM PUGNAc, 2 mM STZ, 10 μM TMG, 40 mM GlcNAc, and Complete protease inhibitors).

    Article Title: Multivalent structure of an ??T cell receptor
    Article Snippet: To allow for linearity and quantitation in the immunofluorescence analyses, a set of six calibration beads displaying predefined amounts of antibodies, QIFIKIT (DAKO), was used to set the optimal signal amplification and compensation levels ( ). .. Transgenic cells were lysed in Brij96, or, alternatively, Nonidet P-40, digitonin, or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, lysis buffer, and the lysate of 2 × 107 cells were immunoprecipitated with the indicated mAb, or species-matched irrelevant mAb, which was covalently coupled to protein A Sepharose beads using dimethylpimelimidate (Sigma). ..

    Co-Immunoprecipitation Assay:

    Article Title: O-GlcNAcylation of SIRT1 enhances its deacetylase activity and promotes cytoprotection under stress
    Article Snippet: Stable cell lines were obtained by retroviral or lentiviral infection and selection with puromycin (1 μg ml−1 ) or hygromycin B (200 μg ml−1 ) for 2 weeks. .. Immunoprecipitation (IP) and CoIP For IP assay, cells were lysed in RIPA buffer (25 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP40, 0.1% SDS, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF, 10 μM PUGNAc [Sigma], 2 mM STZ [Sigma], 10 μM TMG [Sigma], 40 mM GlcNAc [Sigma], and Complete™ protease inhibitors [Roche]). .. For CoIP assay, cells were lysed in NP40 lysis buffer (25 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP40, 1 mM EDTA, 1 mM Na3VO4, 10 mM NaF, 10 μM PUGNAc, 2 mM STZ, 10 μM TMG, 40 mM GlcNAc, and Complete protease inhibitors).

    Lysis:

    Article Title: The Modulation of CD40 Ligand Signaling by Transmembrane CD28 Splice Variant in Human T Cells
    Article Snippet: The Abs specific for human CD28 (L293; BD Biosciences), human CD28 COOH-terminal (sc-1623; Santa Cruz Biotechnology, Inc.), human CD40L (sc-978, sc-9097; Santa Cruz Biotechnology, Inc. and CSA-186; StressGen Biotechnologies), and HA-tag (3F10; Roche and F-7; Santa Cruz Biotechnology, Inc.) were used. .. Cell extracts were prepared using lysis buffer containing 1% Nonidet P-40 (BDH Chemicals) or 1% digitonin (Sigma-Aldrich; reference ). .. To immunoprecipitate CD28i-HA with anti-HA Ab (rat IgG1), protein A/G agarose and protein L agarose bead cocktail (sc-2336; Santa Cruz Biotechnology, Inc.) were used.

    Article Title: Multivalent structure of an ??T cell receptor
    Article Snippet: To allow for linearity and quantitation in the immunofluorescence analyses, a set of six calibration beads displaying predefined amounts of antibodies, QIFIKIT (DAKO), was used to set the optimal signal amplification and compensation levels ( ). .. Transgenic cells were lysed in Brij96, or, alternatively, Nonidet P-40, digitonin, or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, lysis buffer, and the lysate of 2 × 107 cells were immunoprecipitated with the indicated mAb, or species-matched irrelevant mAb, which was covalently coupled to protein A Sepharose beads using dimethylpimelimidate (Sigma). ..

    Transfection:

    Article Title: Complement Component 5a Receptor Oligomerization and Homologous Receptor Down-regulation *
    Article Snippet: Immunoprecipitation and Immunoblotting —HEK293 cells were plated in polylysine-coated 6-well plates and transiently transfected with the appropriate plasmids. .. Two days after transfection, cells were lysed in nondenaturing ice-cold buffer (50 m m Tris, pH 8.0, 150 m m NaCl, 1 m m EDTA, 1% Nonidet P-40, 15% glycerol) containing protease and phosphatase inhibitors (protease inhibitor mixture and phosphatase inhibitor mixture; Sigma). ..

    Protease Inhibitor:

    Article Title: Complement Component 5a Receptor Oligomerization and Homologous Receptor Down-regulation *
    Article Snippet: Immunoprecipitation and Immunoblotting —HEK293 cells were plated in polylysine-coated 6-well plates and transiently transfected with the appropriate plasmids. .. Two days after transfection, cells were lysed in nondenaturing ice-cold buffer (50 m m Tris, pH 8.0, 150 m m NaCl, 1 m m EDTA, 1% Nonidet P-40, 15% glycerol) containing protease and phosphatase inhibitors (protease inhibitor mixture and phosphatase inhibitor mixture; Sigma). ..

    Article Title: Lysosomal targeting of the ABC transporter TAPL is determined by membrane-localized charged residues
    Article Snippet: 8.8 × 106 cells were used for each individual IP. .. Harvested cells were stored at −80 °C, thawed on ice, and solubilized using IP buffer containing 0.5% NP40 (Sigma-Aldrich/Merck) or 1% digitonin (Millipore/Merck) and 1× HP protease inhibitor mix (Serva) for 1 h at 4 °C. .. Supernatant was incubated with antibody-coated beads for 2 h at 4 °C and washed with 3 ml of IP buffer containing 0.05% NP40 or 0.1% digitonin.

    other:

    Article Title: Recruitment of the cross-linked opsonic receptor CD32A (Fc?RIIA) to high-density detergent-resistant membrane domains in human neutrophils
    Article Snippet: iPr2 P -F (di-isopropyl fluorophosphate) was obtained from Helixx Technologies (Scarborough, ON, Canada) and NP40 (Nonidet P40) and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4- d ]pyrimidine (referred to as PP2) were obtained from Calbiochem (San Diego, CA, U.S.A.).

    Transgenic Assay:

    Article Title: Multivalent structure of an ??T cell receptor
    Article Snippet: To allow for linearity and quantitation in the immunofluorescence analyses, a set of six calibration beads displaying predefined amounts of antibodies, QIFIKIT (DAKO), was used to set the optimal signal amplification and compensation levels ( ). .. Transgenic cells were lysed in Brij96, or, alternatively, Nonidet P-40, digitonin, or 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, lysis buffer, and the lysate of 2 × 107 cells were immunoprecipitated with the indicated mAb, or species-matched irrelevant mAb, which was covalently coupled to protein A Sepharose beads using dimethylpimelimidate (Sigma). ..

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    Millipore igepal ca 630
    Giant magnetoresistance biosensor showed higher sensitivity than ELISA for detection of IAV. Swine IAV strain H3N2v or control (mock) were treated with 1% <t>IGEPAL</t> <t>CA-630</t> to disrupt virus particle and used for detection by GMR biosensor and ELISA. (A) Binding curves in real-time on GMR biosensor; (B) Signals averaged over the last 10 data points from different concentrations of IAV and negative control (mock) in GMR biosensor and; (C) Antigen capture ELISA with different concentrations of IAV. Dotted line indicates the cut off value. Error bars represent SEM.
    Igepal Ca 630, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/igepal ca 630/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    igepal ca 630 - by Bioz Stars, 2021-05
    97/100 stars
      Buy from Supplier

    99
    Millipore non ionic detergent triton x 114
    Triton X-114 solubility of different cell-associated and secreted PPAD species.  P .  gingivalis  isolates were cultured in BHI, and the cell and growth medium fractions were separated by centrifugation. Subsequently, both fractions were extracted with Triton X-114. Upon phase separation at 37 °C, proteins in the different detergent-rich (T) and aqueous (A) phases were analyzed by LDS-PAGE and Western blotting with PPAD-specific antibodies. Names of PPAD sorting type I isolates are underlined. Molecular weights of marker proteins and different PPAD species are indicated. The full-length blot is presented in Figure   S1 .
    Non Ionic Detergent Triton X 114, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/non ionic detergent triton x 114/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    non ionic detergent triton x 114 - by Bioz Stars, 2021-05
    99/100 stars
      Buy from Supplier

    Image Search Results


    Giant magnetoresistance biosensor showed higher sensitivity than ELISA for detection of IAV. Swine IAV strain H3N2v or control (mock) were treated with 1% IGEPAL CA-630 to disrupt virus particle and used for detection by GMR biosensor and ELISA. (A) Binding curves in real-time on GMR biosensor; (B) Signals averaged over the last 10 data points from different concentrations of IAV and negative control (mock) in GMR biosensor and; (C) Antigen capture ELISA with different concentrations of IAV. Dotted line indicates the cut off value. Error bars represent SEM.

    Journal: Frontiers in Microbiology

    Article Title: Giant Magnetoresistance-based Biosensor for Detection of Influenza A Virus

    doi: 10.3389/fmicb.2016.00400

    Figure Lengend Snippet: Giant magnetoresistance biosensor showed higher sensitivity than ELISA for detection of IAV. Swine IAV strain H3N2v or control (mock) were treated with 1% IGEPAL CA-630 to disrupt virus particle and used for detection by GMR biosensor and ELISA. (A) Binding curves in real-time on GMR biosensor; (B) Signals averaged over the last 10 data points from different concentrations of IAV and negative control (mock) in GMR biosensor and; (C) Antigen capture ELISA with different concentrations of IAV. Dotted line indicates the cut off value. Error bars represent SEM.

    Article Snippet: For immunoassays the virus was inactivated at 60°C for 1 h. To disrupt the virus particles, the mock and virus preparation were treated with 1% IGEPAL CA-630 (Sigma-Aldrich, Product No. I8896) for 10 min at 37°C.

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Negative Control

    Triton X-114 solubility of different cell-associated and secreted PPAD species.  P .  gingivalis  isolates were cultured in BHI, and the cell and growth medium fractions were separated by centrifugation. Subsequently, both fractions were extracted with Triton X-114. Upon phase separation at 37 °C, proteins in the different detergent-rich (T) and aqueous (A) phases were analyzed by LDS-PAGE and Western blotting with PPAD-specific antibodies. Names of PPAD sorting type I isolates are underlined. Molecular weights of marker proteins and different PPAD species are indicated. The full-length blot is presented in Figure   S1 .

    Journal: Scientific Reports

    Article Title: Dropping anchor: attachment of peptidylarginine deiminase via A-LPS to secreted outer membrane vesicles of Porphyromonas gingivalis

    doi: 10.1038/s41598-018-27223-5

    Figure Lengend Snippet: Triton X-114 solubility of different cell-associated and secreted PPAD species. P . gingivalis isolates were cultured in BHI, and the cell and growth medium fractions were separated by centrifugation. Subsequently, both fractions were extracted with Triton X-114. Upon phase separation at 37 °C, proteins in the different detergent-rich (T) and aqueous (A) phases were analyzed by LDS-PAGE and Western blotting with PPAD-specific antibodies. Names of PPAD sorting type I isolates are underlined. Molecular weights of marker proteins and different PPAD species are indicated. The full-length blot is presented in Figure  S1 .

    Article Snippet: Aqueous two-phase system protein purification To assess modification of the ~75–85-kDa PPAD species with A-LPS, a protein phase separation assay was applied using the non-ionic detergent Triton X-114 (Sigma-Aldrich, St. Louis, USA) .

    Techniques: Solubility, Cell Culture, Centrifugation, Polyacrylamide Gel Electrophoresis, Western Blot, Marker

    A-LPS modification of PPAD in sorting type I and II isolates of  P .  gingivalis . Cells of  P .  gingivalis  sorting type I and II isolates were cultured in BHI and, subsequently, extracted with Triton X-114. Upon phase separation at 37 °C, proteins in the detergent-rich (T) and aqueous (A) phases were analyzed by LDS-PAGE and Western blotting. In panel A immunodetection was performed with an A-LPS-specific monoclonal antibody. Panel B shows a dual-labeling image upon immunodetection with the A-LPS-specific monoclonal antibody (green signal) and a PPAD-specific rabbit antibody (red signal). Bands representing the A-LPS-modified PPAD species of ~75–85-kDa PPAD are boxed in both panels. Names of sorting type I isolates are underlined. Molecular weights of marker proteins are indicated. The full-length blot is presented in Figure   S1 . Please note that the order of ‘A’- and ‘T’-labeled lanes is inversed compared to Fig.   2  as samples were loaded in a different order.

    Journal: Scientific Reports

    Article Title: Dropping anchor: attachment of peptidylarginine deiminase via A-LPS to secreted outer membrane vesicles of Porphyromonas gingivalis

    doi: 10.1038/s41598-018-27223-5

    Figure Lengend Snippet: A-LPS modification of PPAD in sorting type I and II isolates of P . gingivalis . Cells of P . gingivalis sorting type I and II isolates were cultured in BHI and, subsequently, extracted with Triton X-114. Upon phase separation at 37 °C, proteins in the detergent-rich (T) and aqueous (A) phases were analyzed by LDS-PAGE and Western blotting. In panel A immunodetection was performed with an A-LPS-specific monoclonal antibody. Panel B shows a dual-labeling image upon immunodetection with the A-LPS-specific monoclonal antibody (green signal) and a PPAD-specific rabbit antibody (red signal). Bands representing the A-LPS-modified PPAD species of ~75–85-kDa PPAD are boxed in both panels. Names of sorting type I isolates are underlined. Molecular weights of marker proteins are indicated. The full-length blot is presented in Figure  S1 . Please note that the order of ‘A’- and ‘T’-labeled lanes is inversed compared to Fig.  2 as samples were loaded in a different order.

    Article Snippet: Aqueous two-phase system protein purification To assess modification of the ~75–85-kDa PPAD species with A-LPS, a protein phase separation assay was applied using the non-ionic detergent Triton X-114 (Sigma-Aldrich, St. Louis, USA) .

    Techniques: Modification, Cell Culture, Polyacrylamide Gel Electrophoresis, Western Blot, Immunodetection, Labeling, Marker

    Hsp90 is required for TNF-triggered necroptosis. ( a ,  b ) 17AAG treatment inhibits TNF-induced necroptosis. HT29 cells were pretreated with or without different concentrations of 17AAG for 12 h, and then treated with the indicated stimuli for 24 h. Cell images were taken with a Nikon-TE2000 microscope ( a ). Scale bar, 200  μ m. Cell viability was determined by an MTS assay ( b ). Results are the means±S.D. of triplicate measurements. The final concentrations of 20 ng/ml TNF, 100 nM Smac mimetic, 20  μ M z-VAD and 10  μ M necrostatin-1 were used. T, TNF; S, Smac mimetic; Z, z-VAD; Nec-1, necrostatin-1. ( c ) 17AAG treatment reduces the phosphorylation of MLKL. HT29 cells were pretreated with or without different concentrations of 17AAG for 12 h, and then treated with the indicated stimuli for 8 h. The activation of MLKL was analyzed by immunoblotting with a T357/S358 phospho-specific MLKL antibody. Protein levels were detected by western blotting using anti-RIP1, anti-RIP3, anti-MLKL and anti-actin antibodies. ( d ) 17AAG inhibits TNF-induced oligomerization of phosphorylated MLKL. HT29 cells were treated as in ( c ). The cells were harvested and lysed, and non-reducing samples (without  β -mercaptoethanol) of whole-cell lysate were analyzed by immunoblotting with a T357/S358 phospho-specific MLKL antibody. ( e ) 17AAG inhibits plasma membrane translocation of phosphorylated MLKL. HT29 cells were pretreated with or without 125 nM 17AAG for 12 h, and then treated with the indicated stimuli for 8 h. The cells were harvested and then separated into the aqueous phase (Aq) and detergent phase (Det) using Triton X-114 lysis buffer as described in the experimental procedures. The samples were analyzed by western blotting with the indicated antibodies. ( f ) The effect of 17AAG on TNF-induced necrosome formation. Ten-centimetre plates of HT29 cells were pretreated with or without 125 nM 17AAG for 12 h and then stimulated with TNF alone or TSZ for 8 h. Cells were then harvested and whole-cell extracts were immunoprecipitated with anti-RIP3 antibody or anti-IgG antibody and subsequently analyzed by western blotting for the indicated proteins. The asterisks denote nonspecific IgG bands

    Journal: Cell Death & Disease

    Article Title: Hsp90 modulates the stability of MLKL and is required for TNF-induced necroptosis

    doi: 10.1038/cddis.2015.390

    Figure Lengend Snippet: Hsp90 is required for TNF-triggered necroptosis. ( a , b ) 17AAG treatment inhibits TNF-induced necroptosis. HT29 cells were pretreated with or without different concentrations of 17AAG for 12 h, and then treated with the indicated stimuli for 24 h. Cell images were taken with a Nikon-TE2000 microscope ( a ). Scale bar, 200  μ m. Cell viability was determined by an MTS assay ( b ). Results are the means±S.D. of triplicate measurements. The final concentrations of 20 ng/ml TNF, 100 nM Smac mimetic, 20  μ M z-VAD and 10  μ M necrostatin-1 were used. T, TNF; S, Smac mimetic; Z, z-VAD; Nec-1, necrostatin-1. ( c ) 17AAG treatment reduces the phosphorylation of MLKL. HT29 cells were pretreated with or without different concentrations of 17AAG for 12 h, and then treated with the indicated stimuli for 8 h. The activation of MLKL was analyzed by immunoblotting with a T357/S358 phospho-specific MLKL antibody. Protein levels were detected by western blotting using anti-RIP1, anti-RIP3, anti-MLKL and anti-actin antibodies. ( d ) 17AAG inhibits TNF-induced oligomerization of phosphorylated MLKL. HT29 cells were treated as in ( c ). The cells were harvested and lysed, and non-reducing samples (without β -mercaptoethanol) of whole-cell lysate were analyzed by immunoblotting with a T357/S358 phospho-specific MLKL antibody. ( e ) 17AAG inhibits plasma membrane translocation of phosphorylated MLKL. HT29 cells were pretreated with or without 125 nM 17AAG for 12 h, and then treated with the indicated stimuli for 8 h. The cells were harvested and then separated into the aqueous phase (Aq) and detergent phase (Det) using Triton X-114 lysis buffer as described in the experimental procedures. The samples were analyzed by western blotting with the indicated antibodies. ( f ) The effect of 17AAG on TNF-induced necrosome formation. Ten-centimetre plates of HT29 cells were pretreated with or without 125 nM 17AAG for 12 h and then stimulated with TNF alone or TSZ for 8 h. Cells were then harvested and whole-cell extracts were immunoprecipitated with anti-RIP3 antibody or anti-IgG antibody and subsequently analyzed by western blotting for the indicated proteins. The asterisks denote nonspecific IgG bands

    Article Snippet: The following reagents were used: Hsp90 inhibitor 17AAG (Selleckchem, Huston, TX, USA, S1141); z-VAD-fmk (ApexBio, Huston, TX, USA, A1902); Triton X-114 (Sigma-Aldrich, St. Louis, MO, USA); anti-HA (sc-805), anti-Myc (sc-40), anti-RIP3 (sc-374639) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Hsp90 (ab178854), anti-phospho-MLKL (ab187091) (Abcam, Cambridge, MA, USA); anti-MLKL (Merck Millipore, Billerica, MA, USA, MABC604); anti-Flag M2 (Sigma-Aldrich); anti-RIP1 (BD Pharmingen, Franklin Lakes, NJ, USA, 51-6559GR).

    Techniques: Microscopy, MTS Assay, Activation Assay, Western Blot, Translocation Assay, Lysis, Immunoprecipitation

    Coexpression of Hsp90 enhances MLKL-mediated necroptosis. ( a ) The 293T cells were transfected with vector, with Flag-Hsp90 α , with MLKL-Myc alone, or with Flag-Hsp90 α  and MLKL-Myc. After 24 h of transfection, cell images were taken with a Nikon-TE2000 microscope. Scale bar, 100  μ m. ( b ) The 293T cells were transfected as in ( a ). After 24 h of transfection, cell death was quantified by propidium iodide (PI) staining. Cell death data are the means±S.D. of three independent experiments. ( c ) Hsp90 α  increases MLKL oligomerization. The 293T cells were transfected with the indicated plasmids. The cells were harvested 24 h after transfection, and non-reducing samples (without  β -mercaptoethanol) of whole-cell lysate were analyzed by immunoblotting with anti-HA antibody. ( d ) Hsp90 α  increases the plasma membrane translocation of MLKL. The 293T cells were transfected with the indicated expression vectors. After 24 h of transfection, the cells were harvested and lysed in Triton X-114 lysis buffer and then separated into aqueous phase (Aq) and detergent phase (Det) as described in the experimental procedures. The samples were resolved and probed with the indicated antibodies.  β -actin and Cox4 were used as loading controls for soluble protein and membrane protein, respectively

    Journal: Cell Death & Disease

    Article Title: Hsp90 modulates the stability of MLKL and is required for TNF-induced necroptosis

    doi: 10.1038/cddis.2015.390

    Figure Lengend Snippet: Coexpression of Hsp90 enhances MLKL-mediated necroptosis. ( a ) The 293T cells were transfected with vector, with Flag-Hsp90 α , with MLKL-Myc alone, or with Flag-Hsp90 α and MLKL-Myc. After 24 h of transfection, cell images were taken with a Nikon-TE2000 microscope. Scale bar, 100  μ m. ( b ) The 293T cells were transfected as in ( a ). After 24 h of transfection, cell death was quantified by propidium iodide (PI) staining. Cell death data are the means±S.D. of three independent experiments. ( c ) Hsp90 α increases MLKL oligomerization. The 293T cells were transfected with the indicated plasmids. The cells were harvested 24 h after transfection, and non-reducing samples (without β -mercaptoethanol) of whole-cell lysate were analyzed by immunoblotting with anti-HA antibody. ( d ) Hsp90 α increases the plasma membrane translocation of MLKL. The 293T cells were transfected with the indicated expression vectors. After 24 h of transfection, the cells were harvested and lysed in Triton X-114 lysis buffer and then separated into aqueous phase (Aq) and detergent phase (Det) as described in the experimental procedures. The samples were resolved and probed with the indicated antibodies. β -actin and Cox4 were used as loading controls for soluble protein and membrane protein, respectively

    Article Snippet: The following reagents were used: Hsp90 inhibitor 17AAG (Selleckchem, Huston, TX, USA, S1141); z-VAD-fmk (ApexBio, Huston, TX, USA, A1902); Triton X-114 (Sigma-Aldrich, St. Louis, MO, USA); anti-HA (sc-805), anti-Myc (sc-40), anti-RIP3 (sc-374639) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-Hsp90 (ab178854), anti-phospho-MLKL (ab187091) (Abcam, Cambridge, MA, USA); anti-MLKL (Merck Millipore, Billerica, MA, USA, MABC604); anti-Flag M2 (Sigma-Aldrich); anti-RIP1 (BD Pharmingen, Franklin Lakes, NJ, USA, 51-6559GR).

    Techniques: Transfection, Plasmid Preparation, Microscopy, Staining, Translocation Assay, Expressing, Lysis

    CopB and CopB2 partition differently in Triton X-114 extracts. Integral membrane association was tested by extracting  C. trachomatis  L2-infected HeLa cultures with Triton X-114 and separating proteins into aqueous and detergent soluble fractions. (A) Cultures were harvested at 20 h postinfection, and a parallel, mock-treated HeLa culture was harvested as a specificity control. Equivalent quantities of total protein from aqueous (Aq)- and detergent (Det)-soluble samples were resolved by SDS-PAGE in 12% polyacrylamide gels. Immunoblots were probed with anti-MOMP, anti-GroEL, anti-CopN, or anti-IncG as a fractionation control and with antibody specific for CopB or CopB2. Proteins were visualized by probing with secondary antibodies conjugated to alkaline phosphatase and development with NBT-BCIP. (B) HeLa cultures were mock treated or  C. trachomatis  L2 infected at an MOI of 10. Cultures were harvested at 2 h postinfection, extracted with Triton X-114, and probed by immunoblotting with the indicated antibodies. Proteins were visualized by probing with secondary antibodies conjugated to horseradish peroxidase and development with ECL Plus chemiluminescence reagent.

    Journal: Infection and Immunity

    Article Title: Biochemical and Localization Analyses of Putative Type III Secretion Translocator Proteins CopB and CopB2 of Chlamydia trachomatis Reveal Significant Distinctions ▿

    doi: 10.1128/IAI.00159-11

    Figure Lengend Snippet: CopB and CopB2 partition differently in Triton X-114 extracts. Integral membrane association was tested by extracting C. trachomatis L2-infected HeLa cultures with Triton X-114 and separating proteins into aqueous and detergent soluble fractions. (A) Cultures were harvested at 20 h postinfection, and a parallel, mock-treated HeLa culture was harvested as a specificity control. Equivalent quantities of total protein from aqueous (Aq)- and detergent (Det)-soluble samples were resolved by SDS-PAGE in 12% polyacrylamide gels. Immunoblots were probed with anti-MOMP, anti-GroEL, anti-CopN, or anti-IncG as a fractionation control and with antibody specific for CopB or CopB2. Proteins were visualized by probing with secondary antibodies conjugated to alkaline phosphatase and development with NBT-BCIP. (B) HeLa cultures were mock treated or C. trachomatis L2 infected at an MOI of 10. Cultures were harvested at 2 h postinfection, extracted with Triton X-114, and probed by immunoblotting with the indicated antibodies. Proteins were visualized by probing with secondary antibodies conjugated to horseradish peroxidase and development with ECL Plus chemiluminescence reagent.

    Article Snippet: For Triton X-114 extractions, soluble and integral membrane proteins were separated based on solubility in Triton X-114 (Sigma) to examine the partitioning of selected proteins during chlamydial infection.

    Techniques: Infection, SDS Page, Western Blot, Fractionation