Structured Review

Bio-Rad nonidet p 40
Kinetics of [ 3 H]cholesterol binding to purified NPC proteins. ( A  and  B ) Time course at different temperatures. Each reaction, in a final volume of 80 μl of buffer B (pH 6.5) with 0.004% Nonidet P-40, contained 4 pmol of NPC1(NTD)-LVPRGS-His-8-FLAG
Nonidet P 40, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/nonidet p 40/product/Bio-Rad
Average 93 stars, based on 36 article reviews
Price from $9.99 to $1999.99
nonidet p 40 - by Bioz Stars, 2020-11
93/100 stars

Images

1) Product Images from "NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes"

Article Title: NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes

Journal:

doi: 10.1073/pnas.0807328105

Kinetics of [ 3 H]cholesterol binding to purified NPC proteins. ( A  and  B ) Time course at different temperatures. Each reaction, in a final volume of 80 μl of buffer B (pH 6.5) with 0.004% Nonidet P-40, contained 4 pmol of NPC1(NTD)-LVPRGS-His-8-FLAG
Figure Legend Snippet: Kinetics of [ 3 H]cholesterol binding to purified NPC proteins. ( A and B ) Time course at different temperatures. Each reaction, in a final volume of 80 μl of buffer B (pH 6.5) with 0.004% Nonidet P-40, contained 4 pmol of NPC1(NTD)-LVPRGS-His-8-FLAG

Techniques Used: Binding Assay, Purification

2) Product Images from "NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes"

Article Title: NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes

Journal:

doi: 10.1073/pnas.0807328105

Kinetics of [ 3 H]cholesterol binding to purified NPC proteins. ( A  and  B ) Time course at different temperatures. Each reaction, in a final volume of 80 μl of buffer B (pH 6.5) with 0.004% Nonidet P-40, contained 4 pmol of NPC1(NTD)-LVPRGS-His-8-FLAG
Figure Legend Snippet: Kinetics of [ 3 H]cholesterol binding to purified NPC proteins. ( A and B ) Time course at different temperatures. Each reaction, in a final volume of 80 μl of buffer B (pH 6.5) with 0.004% Nonidet P-40, contained 4 pmol of NPC1(NTD)-LVPRGS-His-8-FLAG

Techniques Used: Binding Assay, Purification

Related Articles

Nucleic Acid Electrophoresis:

Article Title: High Prevalence of Seropositivity to a Major Allergen of Anisakis simplex, Ani s 1, in Dyspeptic Patients
Article Snippet: .. The mixture was subjected to electrophoresis in a 16% acrylamide minigel (150 V) under standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis conditions and transferred by diffusion to two nitrocellulose sheets for 18 h. The next day, after being blocked in 3% Nonidet P-40 for 30 min, the membranes were placed on a Mini-Protean II multiscreen (Bio-Rad Laboratories, Hercules, Calif.). .. For specific IgE determination, the different sera were diluted 1:6 in incubation buffer and incubated overnight in each independent chamber.

Silver Staining:

Article Title: The 19S proteasome is directly involved in the regulation of heterochromatin spreading in fission yeast
Article Snippet: .. The proteins were eluted with elution buffer (2 m m ATP, 10 m m Tris-Cl (pH 8.0), 1 m m MgAc, 1 m m imidazole, 3 m m EGTA, 150 m m NaCl, 0.1% Nonidet P-40, 10 m m β-mercaptoethanol, and 10% glycerol), resolved by 4–15% Mini-PROTEAN® TGXTM precast gel (Bio-Rad), and visualized with silver staining. .. The wild-type ORFs encoding Rpt3, Rpt4, and Rpt6 were subjected to random mutagenesis by error-prone PCR using GeneMorph II random mutagenesis kits (Stratagene) according to the manufacturer's protocol.

Article Title: A Gastrointestinal Calpain Complex, G-calpain, Is a Heterodimer of CAPN8 and CAPN9 Calpain Isoforms, Which Play Catalytic and Regulatory Roles, Respectively *
Article Snippet: .. After washing the anti-FLAG-agarose five times with 1 ml of homogenization buffer containing 0.5% Nonidet P-40, the bound proteins were eluted with 0.2 m glycine (pH 2.7), and the eluate was neutralized with Tris-HCl (pH 9.5), mixed with SDS-sample buffer, subjected to SDS-PAGE, and stained with OrioleTM fluorescent gel stain (Bio-Rad), a UV-based fluorescence imaging system that is as sensitive for protein visualization as silver staining. ..

Article Title: Hepatic TRAP80 selectively regulates lipogenic activity of liver X receptor
Article Snippet: .. After binding (20 mM HEPES [pH 7.6], 20% glycerol, 0.2 mM EDTA, 180 mM KCl, 1 mM DTT, 0.05% nonidet P-40, 0.5 mM PMSF, 10 μM T0901317) and washing (20 mM HEPES [pH 7.6], 20% glycerol, 0.2 mM EDTA, 180 mM KCl, 1 mM DTT, 0.1% nonidet P-40, 0.5 mM PMSF), samples were eluted with 0.2% sarkosyl in wash buffer, resolved by 4% to 20% SDS-PAGE (Bio-Rad), and analyzed either by staining with Silver Stain Plus (Bio-Rad) or by immunoblot analysis. ..

Fluorescence:

Article Title: A Gastrointestinal Calpain Complex, G-calpain, Is a Heterodimer of CAPN8 and CAPN9 Calpain Isoforms, Which Play Catalytic and Regulatory Roles, Respectively *
Article Snippet: .. After washing the anti-FLAG-agarose five times with 1 ml of homogenization buffer containing 0.5% Nonidet P-40, the bound proteins were eluted with 0.2 m glycine (pH 2.7), and the eluate was neutralized with Tris-HCl (pH 9.5), mixed with SDS-sample buffer, subjected to SDS-PAGE, and stained with OrioleTM fluorescent gel stain (Bio-Rad), a UV-based fluorescence imaging system that is as sensitive for protein visualization as silver staining. ..

Homogenization:

Article Title: A Gastrointestinal Calpain Complex, G-calpain, Is a Heterodimer of CAPN8 and CAPN9 Calpain Isoforms, Which Play Catalytic and Regulatory Roles, Respectively *
Article Snippet: .. After washing the anti-FLAG-agarose five times with 1 ml of homogenization buffer containing 0.5% Nonidet P-40, the bound proteins were eluted with 0.2 m glycine (pH 2.7), and the eluate was neutralized with Tris-HCl (pH 9.5), mixed with SDS-sample buffer, subjected to SDS-PAGE, and stained with OrioleTM fluorescent gel stain (Bio-Rad), a UV-based fluorescence imaging system that is as sensitive for protein visualization as silver staining. ..

Electrophoresis:

Article Title: High Prevalence of Seropositivity to a Major Allergen of Anisakis simplex, Ani s 1, in Dyspeptic Patients
Article Snippet: .. The mixture was subjected to electrophoresis in a 16% acrylamide minigel (150 V) under standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis conditions and transferred by diffusion to two nitrocellulose sheets for 18 h. The next day, after being blocked in 3% Nonidet P-40 for 30 min, the membranes were placed on a Mini-Protean II multiscreen (Bio-Rad Laboratories, Hercules, Calif.). .. For specific IgE determination, the different sera were diluted 1:6 in incubation buffer and incubated overnight in each independent chamber.

Immunoprecipitation:

Article Title: The FBXL10/KDM2B Scaffolding Protein Associates with Novel Polycomb Repressive Complex-1 to Regulate Adipogenesis *
Article Snippet: .. Supernatants were collected, and buffer was exchanged to 50 m m Tris-HCl (pH 8.0), 100 m m KCl, 0.1 m m EDTA, 5% glycerol, 0.1% Nonidet P-40 by Econo-Pac 10DG (Bio-Rad), ultrafiltrated by Amicon Ultra-4 MWCO 30K (Millipore), and used for immunoprecipitation. .. The samples were incubated with control IgG or anti-V5 epitope antibody cross-linked with Dynabeads® protein G (Invitrogen) and rotated for 18 h at 4 °C.

Incubation:

Article Title: NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes
Article Snippet: .. After incubation for 30 min at 4°C or 37°C, each reaction mixture was diluted with 500 μl of buffer A supplemented with 0.004% Nonidet P-40 and loaded onto a 2-ml column (Bio-Rad) packed with 0.3 ml of Ni-NTA-agarose beads (Qiagen) that had been preequilibrated with buffer A with 0.004% Nonidet P-40. ..

Diffusion-based Assay:

Article Title: High Prevalence of Seropositivity to a Major Allergen of Anisakis simplex, Ani s 1, in Dyspeptic Patients
Article Snippet: .. The mixture was subjected to electrophoresis in a 16% acrylamide minigel (150 V) under standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis conditions and transferred by diffusion to two nitrocellulose sheets for 18 h. The next day, after being blocked in 3% Nonidet P-40 for 30 min, the membranes were placed on a Mini-Protean II multiscreen (Bio-Rad Laboratories, Hercules, Calif.). .. For specific IgE determination, the different sera were diluted 1:6 in incubation buffer and incubated overnight in each independent chamber.

Imaging:

Article Title: A Gastrointestinal Calpain Complex, G-calpain, Is a Heterodimer of CAPN8 and CAPN9 Calpain Isoforms, Which Play Catalytic and Regulatory Roles, Respectively *
Article Snippet: .. After washing the anti-FLAG-agarose five times with 1 ml of homogenization buffer containing 0.5% Nonidet P-40, the bound proteins were eluted with 0.2 m glycine (pH 2.7), and the eluate was neutralized with Tris-HCl (pH 9.5), mixed with SDS-sample buffer, subjected to SDS-PAGE, and stained with OrioleTM fluorescent gel stain (Bio-Rad), a UV-based fluorescence imaging system that is as sensitive for protein visualization as silver staining. ..

Staining:

Article Title: A Gastrointestinal Calpain Complex, G-calpain, Is a Heterodimer of CAPN8 and CAPN9 Calpain Isoforms, Which Play Catalytic and Regulatory Roles, Respectively *
Article Snippet: .. After washing the anti-FLAG-agarose five times with 1 ml of homogenization buffer containing 0.5% Nonidet P-40, the bound proteins were eluted with 0.2 m glycine (pH 2.7), and the eluate was neutralized with Tris-HCl (pH 9.5), mixed with SDS-sample buffer, subjected to SDS-PAGE, and stained with OrioleTM fluorescent gel stain (Bio-Rad), a UV-based fluorescence imaging system that is as sensitive for protein visualization as silver staining. ..

Article Title: Hepatic TRAP80 selectively regulates lipogenic activity of liver X receptor
Article Snippet: .. After binding (20 mM HEPES [pH 7.6], 20% glycerol, 0.2 mM EDTA, 180 mM KCl, 1 mM DTT, 0.05% nonidet P-40, 0.5 mM PMSF, 10 μM T0901317) and washing (20 mM HEPES [pH 7.6], 20% glycerol, 0.2 mM EDTA, 180 mM KCl, 1 mM DTT, 0.1% nonidet P-40, 0.5 mM PMSF), samples were eluted with 0.2% sarkosyl in wash buffer, resolved by 4% to 20% SDS-PAGE (Bio-Rad), and analyzed either by staining with Silver Stain Plus (Bio-Rad) or by immunoblot analysis. ..

Lysis:

Article Title: Identification of Differentially Regulated Secretome Components During Skeletal Myogenesis *
Article Snippet: .. The supernatant was discarded and the remaining pellet was resuspended in 200 μl lysis buffer composed of 50 m m Tris-HCl (Bioshop), 150 m m NaCl (Bioshop), 0.5% Nonidet P-40 (BioRad), 2 m m EDTA (Bioshop), 100 m m NaF (Sigma), 10 m m Na2 HPO4 (Bioshop), 1 m m Na3 VO4 (Sigma), 1 m m PMSF (Bioshop), 1 μg/ml leupeptin (Bioshop), 1 μg/ml aprotinin (Bioshop), and 1 μg/ml pepstatin A (Bioshop). .. To examine the expression pattern of the secretome during myogenesis, CM were collected from light-labeled MBs and heavy-labeled MTs cultured in serum-free DM for 24 h and 120 h, respectively (see Step 2 of ).

Binding Assay:

Article Title: Hepatic TRAP80 selectively regulates lipogenic activity of liver X receptor
Article Snippet: .. After binding (20 mM HEPES [pH 7.6], 20% glycerol, 0.2 mM EDTA, 180 mM KCl, 1 mM DTT, 0.05% nonidet P-40, 0.5 mM PMSF, 10 μM T0901317) and washing (20 mM HEPES [pH 7.6], 20% glycerol, 0.2 mM EDTA, 180 mM KCl, 1 mM DTT, 0.1% nonidet P-40, 0.5 mM PMSF), samples were eluted with 0.2% sarkosyl in wash buffer, resolved by 4% to 20% SDS-PAGE (Bio-Rad), and analyzed either by staining with Silver Stain Plus (Bio-Rad) or by immunoblot analysis. ..

SDS Page:

Article Title: A Gastrointestinal Calpain Complex, G-calpain, Is a Heterodimer of CAPN8 and CAPN9 Calpain Isoforms, Which Play Catalytic and Regulatory Roles, Respectively *
Article Snippet: .. After washing the anti-FLAG-agarose five times with 1 ml of homogenization buffer containing 0.5% Nonidet P-40, the bound proteins were eluted with 0.2 m glycine (pH 2.7), and the eluate was neutralized with Tris-HCl (pH 9.5), mixed with SDS-sample buffer, subjected to SDS-PAGE, and stained with OrioleTM fluorescent gel stain (Bio-Rad), a UV-based fluorescence imaging system that is as sensitive for protein visualization as silver staining. ..

Article Title: Hepatic TRAP80 selectively regulates lipogenic activity of liver X receptor
Article Snippet: .. After binding (20 mM HEPES [pH 7.6], 20% glycerol, 0.2 mM EDTA, 180 mM KCl, 1 mM DTT, 0.05% nonidet P-40, 0.5 mM PMSF, 10 μM T0901317) and washing (20 mM HEPES [pH 7.6], 20% glycerol, 0.2 mM EDTA, 180 mM KCl, 1 mM DTT, 0.1% nonidet P-40, 0.5 mM PMSF), samples were eluted with 0.2% sarkosyl in wash buffer, resolved by 4% to 20% SDS-PAGE (Bio-Rad), and analyzed either by staining with Silver Stain Plus (Bio-Rad) or by immunoblot analysis. ..

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 92
    Bio-Rad np 40 buffer
    HCMV gH/gL/gO and gH/gL/UL128-131 do not significantly complex with gB in virions. HCMV BADrUL131 extracellular virus particles were partially purified from cell supernatants harvested10 days post -infection then solubilized in 1% <t>NP-40</t> and insoluble proteins removed by centrifugation. (A) Proteins from virion extracts were IP’d using rabbit polyclonal anti-peptide antibodies specific for UL130 or a gM-specific MAb as a negative control and the IP’d proteins separated by SDS-PAGE and analyzed by western blot with gH-specific MAb AP86 or gB-specific MAb 15H7. (B) Proteins were IP’d from detergent extracts of HCMV virions with rabbit polyclonal antibodies specific for UL130, rabbit polyclonal anti-peptide serum specific for gO (TBgO) [ 23 ] or with a gM-specific MAb and the IP’s analyzed by western blot as described above with the gH-specific MAb AP86. (C) Detergent extracts of extracellular HCMV virus particles were solubilized then extracts IP’d with anti-gM MAb or anti-gB MAb 15H7 (as indicated along the top of the panel). The precipitated proteins were analyzed by western blot with anti-gH MAb AP86, rabbit anti-gO (TBgO) sera, rabbit anti-UL130 sera, or a gM-specific MAb (as indicated along the right side of the panel). (D) IP-western blot analyses of solubilized BADrUL131 extracellular virions as described for panel C, except that the membrane blotted for gH from the gB IP was exposed along side a linear range of input samples derived from a gH/gL expressing cell lysate. (E) Linear curve of the gH signal generated after exposure of the membrane containing the increasing doses of the gH-gL expressing lysate. The y-axis indicates the relative density of the protein bands and the x-axis indicates the amount of lysate loaded into each well. (F) Shown is the exposure of the western blot containing the increasing amounts of the gH-gL expressing lysate. The amount to gH/gL expressing lysate loaded into each well is indicated below the panel. (G) Western blot analyses as described for panel D except IP’s were performed with solubilized extracellular virions derived from the clinical strain TR. (H) Linear curve of the gH signal containing the increasing doses of the gH-gL expressing lysate as described for panel E. (I) Shown is the exposure of the western blot containing the increasing amounts of the gH-gL expressing lysate. The amount to gH/gL expressing lysate loaded into each well is indicated below the panel. To assess the relative quantity of IP’d proteins, we compared the signal intensities of the IP’d proteins to the input protein signal intensities. Analysis was performed using ImageJ software (panel C) or Image Studio software (Licor) for panels 7D-7I. For all blots, input refers to 5% of the virion lysate loaded directly into gels. The percent of the total protein IP’d compared with the total in the virion extract is shown under each lane. Molecular mass (MW) markers are indicated on the left. All samples were analyzed by SDS-PAGE under reducing conditions with the exception of samples involving the detection of gO, which required that that SDS-PAGE be performed under non-reducing conditions, thus the signal for gO represents the gH/gL/gO disulfide linked > 250 kDa trimer. ND indicates not detected.
    Np 40 Buffer, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/np 40 buffer/product/Bio-Rad
    Average 92 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    np 40 buffer - by Bioz Stars, 2020-11
    92/100 stars
      Buy from Supplier

    93
    Bio-Rad nonidet p 40
    Kinetics of [ 3 H]cholesterol binding to purified NPC proteins. ( A  and  B ) Time course at different temperatures. Each reaction, in a final volume of 80 μl of buffer B (pH 6.5) with 0.004% Nonidet P-40, contained 4 pmol of NPC1(NTD)-LVPRGS-His-8-FLAG
    Nonidet P 40, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nonidet p 40/product/Bio-Rad
    Average 93 stars, based on 36 article reviews
    Price from $9.99 to $1999.99
    nonidet p 40 - by Bioz Stars, 2020-11
    93/100 stars
      Buy from Supplier

    85
    Bio-Rad triton x 114 extracts
    Segregation of Msp and CTLP in Triton X-114 extracts, as shown by Western immunoblot analysis. Samples were not heated before electrophoresis. The blots were probed with a mixture of antisera raised against Msp and CTLP. Lanes: 1, crude Triton X-114 extract; 2, extract following high-speed centrifugation; 3, aqueous phase of Triton X-114 extract; 4, detergent phase of Triton X-114 extract. Arrows: Msp oligomers with an apparent molecular mass of approximately 150 kDa; CTLP doublet at approximately 95 kDa.
    Triton X 114 Extracts, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/triton x 114 extracts/product/Bio-Rad
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    triton x 114 extracts - by Bioz Stars, 2020-11
    85/100 stars
      Buy from Supplier

    85
    Bio-Rad temperature dependent triton x 114 phase partitioning
    MSP2(P44) MAb 20B4-screened Western blot of  A. phagocytophilum  HGE1 whole-cell lysate and Triton X-114-partitioned hydrophobic and hydrophilic liquid fractions. Ten micrograms each of  A. phagocytophilum  HGE1 whole-cell lysate and the hydrophobic liquid
    Temperature Dependent Triton X 114 Phase Partitioning, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/temperature dependent triton x 114 phase partitioning/product/Bio-Rad
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    temperature dependent triton x 114 phase partitioning - by Bioz Stars, 2020-11
    85/100 stars
      Buy from Supplier

    Image Search Results


    HCMV gH/gL/gO and gH/gL/UL128-131 do not significantly complex with gB in virions. HCMV BADrUL131 extracellular virus particles were partially purified from cell supernatants harvested10 days post -infection then solubilized in 1% NP-40 and insoluble proteins removed by centrifugation. (A) Proteins from virion extracts were IP’d using rabbit polyclonal anti-peptide antibodies specific for UL130 or a gM-specific MAb as a negative control and the IP’d proteins separated by SDS-PAGE and analyzed by western blot with gH-specific MAb AP86 or gB-specific MAb 15H7. (B) Proteins were IP’d from detergent extracts of HCMV virions with rabbit polyclonal antibodies specific for UL130, rabbit polyclonal anti-peptide serum specific for gO (TBgO) [ 23 ] or with a gM-specific MAb and the IP’s analyzed by western blot as described above with the gH-specific MAb AP86. (C) Detergent extracts of extracellular HCMV virus particles were solubilized then extracts IP’d with anti-gM MAb or anti-gB MAb 15H7 (as indicated along the top of the panel). The precipitated proteins were analyzed by western blot with anti-gH MAb AP86, rabbit anti-gO (TBgO) sera, rabbit anti-UL130 sera, or a gM-specific MAb (as indicated along the right side of the panel). (D) IP-western blot analyses of solubilized BADrUL131 extracellular virions as described for panel C, except that the membrane blotted for gH from the gB IP was exposed along side a linear range of input samples derived from a gH/gL expressing cell lysate. (E) Linear curve of the gH signal generated after exposure of the membrane containing the increasing doses of the gH-gL expressing lysate. The y-axis indicates the relative density of the protein bands and the x-axis indicates the amount of lysate loaded into each well. (F) Shown is the exposure of the western blot containing the increasing amounts of the gH-gL expressing lysate. The amount to gH/gL expressing lysate loaded into each well is indicated below the panel. (G) Western blot analyses as described for panel D except IP’s were performed with solubilized extracellular virions derived from the clinical strain TR. (H) Linear curve of the gH signal containing the increasing doses of the gH-gL expressing lysate as described for panel E. (I) Shown is the exposure of the western blot containing the increasing amounts of the gH-gL expressing lysate. The amount to gH/gL expressing lysate loaded into each well is indicated below the panel. To assess the relative quantity of IP’d proteins, we compared the signal intensities of the IP’d proteins to the input protein signal intensities. Analysis was performed using ImageJ software (panel C) or Image Studio software (Licor) for panels 7D-7I. For all blots, input refers to 5% of the virion lysate loaded directly into gels. The percent of the total protein IP’d compared with the total in the virion extract is shown under each lane. Molecular mass (MW) markers are indicated on the left. All samples were analyzed by SDS-PAGE under reducing conditions with the exception of samples involving the detection of gO, which required that that SDS-PAGE be performed under non-reducing conditions, thus the signal for gO represents the gH/gL/gO disulfide linked > 250 kDa trimer. ND indicates not detected.

    Journal: PLoS Pathogens

    Article Title: Human Cytomegalovirus gH/gL Forms a Stable Complex with the Fusion Protein gB in Virions

    doi: 10.1371/journal.ppat.1005564

    Figure Lengend Snippet: HCMV gH/gL/gO and gH/gL/UL128-131 do not significantly complex with gB in virions. HCMV BADrUL131 extracellular virus particles were partially purified from cell supernatants harvested10 days post -infection then solubilized in 1% NP-40 and insoluble proteins removed by centrifugation. (A) Proteins from virion extracts were IP’d using rabbit polyclonal anti-peptide antibodies specific for UL130 or a gM-specific MAb as a negative control and the IP’d proteins separated by SDS-PAGE and analyzed by western blot with gH-specific MAb AP86 or gB-specific MAb 15H7. (B) Proteins were IP’d from detergent extracts of HCMV virions with rabbit polyclonal antibodies specific for UL130, rabbit polyclonal anti-peptide serum specific for gO (TBgO) [ 23 ] or with a gM-specific MAb and the IP’s analyzed by western blot as described above with the gH-specific MAb AP86. (C) Detergent extracts of extracellular HCMV virus particles were solubilized then extracts IP’d with anti-gM MAb or anti-gB MAb 15H7 (as indicated along the top of the panel). The precipitated proteins were analyzed by western blot with anti-gH MAb AP86, rabbit anti-gO (TBgO) sera, rabbit anti-UL130 sera, or a gM-specific MAb (as indicated along the right side of the panel). (D) IP-western blot analyses of solubilized BADrUL131 extracellular virions as described for panel C, except that the membrane blotted for gH from the gB IP was exposed along side a linear range of input samples derived from a gH/gL expressing cell lysate. (E) Linear curve of the gH signal generated after exposure of the membrane containing the increasing doses of the gH-gL expressing lysate. The y-axis indicates the relative density of the protein bands and the x-axis indicates the amount of lysate loaded into each well. (F) Shown is the exposure of the western blot containing the increasing amounts of the gH-gL expressing lysate. The amount to gH/gL expressing lysate loaded into each well is indicated below the panel. (G) Western blot analyses as described for panel D except IP’s were performed with solubilized extracellular virions derived from the clinical strain TR. (H) Linear curve of the gH signal containing the increasing doses of the gH-gL expressing lysate as described for panel E. (I) Shown is the exposure of the western blot containing the increasing amounts of the gH-gL expressing lysate. The amount to gH/gL expressing lysate loaded into each well is indicated below the panel. To assess the relative quantity of IP’d proteins, we compared the signal intensities of the IP’d proteins to the input protein signal intensities. Analysis was performed using ImageJ software (panel C) or Image Studio software (Licor) for panels 7D-7I. For all blots, input refers to 5% of the virion lysate loaded directly into gels. The percent of the total protein IP’d compared with the total in the virion extract is shown under each lane. Molecular mass (MW) markers are indicated on the left. All samples were analyzed by SDS-PAGE under reducing conditions with the exception of samples involving the detection of gO, which required that that SDS-PAGE be performed under non-reducing conditions, thus the signal for gO represents the gH/gL/gO disulfide linked > 250 kDa trimer. ND indicates not detected.

    Article Snippet: Cells were solubilized in 1% NP-40 buffer and the total protein concentration of the lysate was determined using the Bio-Rad protein assay.

    Techniques: Purification, Infection, Centrifugation, Negative Control, SDS Page, Western Blot, Derivative Assay, Expressing, Generated, Software

    Detection of gB-gH/gL complexes following IP from radiolabeled Ad-transduced cells. ARPE-19 cells were transduced with Ad vectors expressing HCMV glycoproteins: HCMV strain TR or strain AD169 gB, TR gH/gL or a negative control, Ad-tet-trans (tet), as indicated at the top part of each panel. At 20 hrs post-transduction, the cells were radiolabeled with 35 S-cysteine-methionine for 4 hrs. The cells were lysed with IP buffer containing 1% NP-40 and the proteins immunoprecipitated (IP’d) then analyzed by SDS-PAGE under reducing conditions. (A) Lysates were IP’d with anti-gH MAbs: 14-4b or AP86. (B) Lysates were IP’d with rabbit anti-peptide sera specific for gL in the absence of the gL peptide used to produce the antibodies (left panel) or with that peptide present in the IP (right panel). (C) Lysates were IP’d with the human anti-gB MAb 758. Molecular mass (MW) markers are indicated on the left.

    Journal: PLoS Pathogens

    Article Title: Human Cytomegalovirus gH/gL Forms a Stable Complex with the Fusion Protein gB in Virions

    doi: 10.1371/journal.ppat.1005564

    Figure Lengend Snippet: Detection of gB-gH/gL complexes following IP from radiolabeled Ad-transduced cells. ARPE-19 cells were transduced with Ad vectors expressing HCMV glycoproteins: HCMV strain TR or strain AD169 gB, TR gH/gL or a negative control, Ad-tet-trans (tet), as indicated at the top part of each panel. At 20 hrs post-transduction, the cells were radiolabeled with 35 S-cysteine-methionine for 4 hrs. The cells were lysed with IP buffer containing 1% NP-40 and the proteins immunoprecipitated (IP’d) then analyzed by SDS-PAGE under reducing conditions. (A) Lysates were IP’d with anti-gH MAbs: 14-4b or AP86. (B) Lysates were IP’d with rabbit anti-peptide sera specific for gL in the absence of the gL peptide used to produce the antibodies (left panel) or with that peptide present in the IP (right panel). (C) Lysates were IP’d with the human anti-gB MAb 758. Molecular mass (MW) markers are indicated on the left.

    Article Snippet: Cells were solubilized in 1% NP-40 buffer and the total protein concentration of the lysate was determined using the Bio-Rad protein assay.

    Techniques: Transduction, Expressing, Negative Control, Immunoprecipitation, SDS Page

    HCMV gB-gH/gL complexes detected by IP-western blots. ARPE-19 or MRC-5 cells were transduced with Ad vectors expressing HCMV gB, gH/gL, both gB and gH/gL, or with Ad-tet-trans (tet, as a negative control) as indicated at the top part of each panel. After 20 hrs the cells were lysed in IP buffer containing 1% NP-40. (A) Proteins were IP’d from ARPE-19 or MRC-5 cell extracts with anti-gH MAb 14-4b, separated by SDS-PAGE under reducing conditions, transferred to membranes, and then analyzed by western blot using rabbit polyclonal sera specific to gB or anti-gH MAb AP86. (B) Proteins from ARPE-19 cell lysates were IP’d with anti-gB MAbs 9C1, 13H10, or 15H7 and the IPs analyzed by western blot as described above using anti-gH AP86. Input represents 5% of the extract loaded directly onto gels then blotted. (C) ARPE-19 cells were transfected with an Ad vector expressing gB with a C-terminal FLAG epitope tag or wild type gB and co-transduced with Ad vectors to express gH/gL as indicated along the top of the panel. Proteins were IP’d with an anti-FLAG antibody and analyzed by western blot as described above with rabbit polyclonal gB-specific serum or anti-gH MAb AP-86 to detect gB and gH, respectively. The percent of gB and gH/gL that was co-IP in these experiments was quantified using NIH ImageJ software by comparing the relative band intensities from the IP’d proteins to the 5% input and are indicated under the lanes. Molecular mass (MW) markers are indicated on the left.

    Journal: PLoS Pathogens

    Article Title: Human Cytomegalovirus gH/gL Forms a Stable Complex with the Fusion Protein gB in Virions

    doi: 10.1371/journal.ppat.1005564

    Figure Lengend Snippet: HCMV gB-gH/gL complexes detected by IP-western blots. ARPE-19 or MRC-5 cells were transduced with Ad vectors expressing HCMV gB, gH/gL, both gB and gH/gL, or with Ad-tet-trans (tet, as a negative control) as indicated at the top part of each panel. After 20 hrs the cells were lysed in IP buffer containing 1% NP-40. (A) Proteins were IP’d from ARPE-19 or MRC-5 cell extracts with anti-gH MAb 14-4b, separated by SDS-PAGE under reducing conditions, transferred to membranes, and then analyzed by western blot using rabbit polyclonal sera specific to gB or anti-gH MAb AP86. (B) Proteins from ARPE-19 cell lysates were IP’d with anti-gB MAbs 9C1, 13H10, or 15H7 and the IPs analyzed by western blot as described above using anti-gH AP86. Input represents 5% of the extract loaded directly onto gels then blotted. (C) ARPE-19 cells were transfected with an Ad vector expressing gB with a C-terminal FLAG epitope tag or wild type gB and co-transduced with Ad vectors to express gH/gL as indicated along the top of the panel. Proteins were IP’d with an anti-FLAG antibody and analyzed by western blot as described above with rabbit polyclonal gB-specific serum or anti-gH MAb AP-86 to detect gB and gH, respectively. The percent of gB and gH/gL that was co-IP in these experiments was quantified using NIH ImageJ software by comparing the relative band intensities from the IP’d proteins to the 5% input and are indicated under the lanes. Molecular mass (MW) markers are indicated on the left.

    Article Snippet: Cells were solubilized in 1% NP-40 buffer and the total protein concentration of the lysate was determined using the Bio-Rad protein assay.

    Techniques: Western Blot, Transduction, Expressing, Negative Control, SDS Page, Transfection, Plasmid Preparation, FLAG-tag, Co-Immunoprecipitation Assay, Software

    Different detergents and dilution of cell extracts does not destabilize gB-gH/gL complexes. (A) ARPE-19 cells were transduced with Ad vectors expressing gB, gB and gH/gL, or gH/gL and GFP for 20 hrs then radiolabeled for 4 hrs. Cell extract were made using IP buffer containing 1% NP-40. Some of each extract was then diluted with 4 times the volume of the same buffer (4X). Proteins were precipitated from cell extracts using either anti-gB MAb 27–156 or anti-gH MAb 14-4b then proteins analyzed by SDS-PAGE under reducing conditions. (B) ARPE-19 cells were transduced as described for panel A and extracts were prepared with IP buffer containing 1% digitonin. IP’s were performed with either anti-gB MAb 27–156 or anti-gH MAb 14-4b with undiluted samples (1X) or after samples were diluted with 4 times the volume (4X) of IP buffer containing 1% digitonin. (C) IPs were performed as described for panel B except cell lysates were prepared and diluted with IP buffer containing 1% octyl glucoside. The antibodies used for the IPs and the Ad vectors used to transduce cells are indicated above each panel. The detergents used in the IPs and whether samples were used neat or diluted is indicated below each panel. The positions of gB and gH/gL are indicated on the left side of the panels and molecular mass markers (MW) are indicated on the right.

    Journal: PLoS Pathogens

    Article Title: Human Cytomegalovirus gH/gL Forms a Stable Complex with the Fusion Protein gB in Virions

    doi: 10.1371/journal.ppat.1005564

    Figure Lengend Snippet: Different detergents and dilution of cell extracts does not destabilize gB-gH/gL complexes. (A) ARPE-19 cells were transduced with Ad vectors expressing gB, gB and gH/gL, or gH/gL and GFP for 20 hrs then radiolabeled for 4 hrs. Cell extract were made using IP buffer containing 1% NP-40. Some of each extract was then diluted with 4 times the volume of the same buffer (4X). Proteins were precipitated from cell extracts using either anti-gB MAb 27–156 or anti-gH MAb 14-4b then proteins analyzed by SDS-PAGE under reducing conditions. (B) ARPE-19 cells were transduced as described for panel A and extracts were prepared with IP buffer containing 1% digitonin. IP’s were performed with either anti-gB MAb 27–156 or anti-gH MAb 14-4b with undiluted samples (1X) or after samples were diluted with 4 times the volume (4X) of IP buffer containing 1% digitonin. (C) IPs were performed as described for panel B except cell lysates were prepared and diluted with IP buffer containing 1% octyl glucoside. The antibodies used for the IPs and the Ad vectors used to transduce cells are indicated above each panel. The detergents used in the IPs and whether samples were used neat or diluted is indicated below each panel. The positions of gB and gH/gL are indicated on the left side of the panels and molecular mass markers (MW) are indicated on the right.

    Article Snippet: Cells were solubilized in 1% NP-40 buffer and the total protein concentration of the lysate was determined using the Bio-Rad protein assay.

    Techniques: Transduction, Expressing, SDS Page

    gB-gH/gL complexes form early after synthesis and are stable. ARPE-19 cells were transduced with Ad vectors expressing either gB or gH/gL alone or with a combination of both gB and gH/gL. After 24 hrs, the cells were radiolabeled for 15 min. with 35 S-methionine/cysteine then 1% NP-40 cell extracts made immediately (P) or the radiolabeled cells incubated in chase media with excess non-radioactive methionine and cysteine, i.e. the label chased (C) for 60, 120, or 240 min. After these chase periods, cell extracts were made immediately and proteins IP’d with anti-gH MAb 14-4b (IP: gH) or anti-gB rabbit serum (IP:gB) and analyzed by SDS-PAGE under reducing conditions. Molecular mass (MW) markers are indicated on the right.

    Journal: PLoS Pathogens

    Article Title: Human Cytomegalovirus gH/gL Forms a Stable Complex with the Fusion Protein gB in Virions

    doi: 10.1371/journal.ppat.1005564

    Figure Lengend Snippet: gB-gH/gL complexes form early after synthesis and are stable. ARPE-19 cells were transduced with Ad vectors expressing either gB or gH/gL alone or with a combination of both gB and gH/gL. After 24 hrs, the cells were radiolabeled for 15 min. with 35 S-methionine/cysteine then 1% NP-40 cell extracts made immediately (P) or the radiolabeled cells incubated in chase media with excess non-radioactive methionine and cysteine, i.e. the label chased (C) for 60, 120, or 240 min. After these chase periods, cell extracts were made immediately and proteins IP’d with anti-gH MAb 14-4b (IP: gH) or anti-gB rabbit serum (IP:gB) and analyzed by SDS-PAGE under reducing conditions. Molecular mass (MW) markers are indicated on the right.

    Article Snippet: Cells were solubilized in 1% NP-40 buffer and the total protein concentration of the lysate was determined using the Bio-Rad protein assay.

    Techniques: Transduction, Expressing, Incubation, SDS Page

    gB-gH/gL complexes in HCMV-infected cells and extracellular virions. (A) Human fibroblasts were infected with HCMV BADrUL131 for 3 days then cells were pelleted and cell extracts made using 1% NP-40. gH/gL was IP’d using MAb 14-4b or cell extracts incubated with an irrelevant MAb specific for FLAG (no FLAG epitope was present in gH/gL). The IP’d proteins were then subjected to SDS-PAGE under reducing conditions and analyzed by western blot using rabbit gB-specific antiserum (gB blot) or anti-gH MAb AP86 (gH blot). (B) HCMV extracellular particles partially purified from 10-day HCMV infected fibroblast culture supernatants were solubilized in 1% NP-40 and the proteins IP’d with MAb anti-FLAG (irrelevant MAb), anti-gH 14-4b, or an anti-gM MAb. The IP’d proteins were analyzed by western blot as described above using rabbit anti-gB polyclonal sera, anti-gH MAb AP86, or a gM MAb. (C) Proteins from solubilized extracellular virus particles were IP’d with rabbit anti-gL peptide serum (IP:gL), a control antibody to HSV-1 gB (IP:R68) or rabbit anti-gL peptide serum alone with no HCMV lysate (IP:gL no lysate) to control for cross reactivity of the IgG in the serum with anti-rabbit secondary antibodies. The proteins were analyzed by western blot and membranes were probed with anti-HCMV gB MAb 27–156 (gB blot, upper panel) or with rabbit anti-gH peptide serum (gH blot, lower panel). Migration of the rabbit IgG which was picked up in the in the gB blot is indicated by the asterisk. In panels A, B, and C, input represents 5% of the total amount of lysate. (D) An equal amount of cell-associated virus stock (cell) or virus stock purified from cell supernatants (virions) was separated by SDS-PAGE, transferred to membranes and blotted with anti-HCMV gB MAb 15H7 or a polyclonal rabbit antibody to calnexin. Molecular mass (MW) markers are indicated on the right.

    Journal: PLoS Pathogens

    Article Title: Human Cytomegalovirus gH/gL Forms a Stable Complex with the Fusion Protein gB in Virions

    doi: 10.1371/journal.ppat.1005564

    Figure Lengend Snippet: gB-gH/gL complexes in HCMV-infected cells and extracellular virions. (A) Human fibroblasts were infected with HCMV BADrUL131 for 3 days then cells were pelleted and cell extracts made using 1% NP-40. gH/gL was IP’d using MAb 14-4b or cell extracts incubated with an irrelevant MAb specific for FLAG (no FLAG epitope was present in gH/gL). The IP’d proteins were then subjected to SDS-PAGE under reducing conditions and analyzed by western blot using rabbit gB-specific antiserum (gB blot) or anti-gH MAb AP86 (gH blot). (B) HCMV extracellular particles partially purified from 10-day HCMV infected fibroblast culture supernatants were solubilized in 1% NP-40 and the proteins IP’d with MAb anti-FLAG (irrelevant MAb), anti-gH 14-4b, or an anti-gM MAb. The IP’d proteins were analyzed by western blot as described above using rabbit anti-gB polyclonal sera, anti-gH MAb AP86, or a gM MAb. (C) Proteins from solubilized extracellular virus particles were IP’d with rabbit anti-gL peptide serum (IP:gL), a control antibody to HSV-1 gB (IP:R68) or rabbit anti-gL peptide serum alone with no HCMV lysate (IP:gL no lysate) to control for cross reactivity of the IgG in the serum with anti-rabbit secondary antibodies. The proteins were analyzed by western blot and membranes were probed with anti-HCMV gB MAb 27–156 (gB blot, upper panel) or with rabbit anti-gH peptide serum (gH blot, lower panel). Migration of the rabbit IgG which was picked up in the in the gB blot is indicated by the asterisk. In panels A, B, and C, input represents 5% of the total amount of lysate. (D) An equal amount of cell-associated virus stock (cell) or virus stock purified from cell supernatants (virions) was separated by SDS-PAGE, transferred to membranes and blotted with anti-HCMV gB MAb 15H7 or a polyclonal rabbit antibody to calnexin. Molecular mass (MW) markers are indicated on the right.

    Article Snippet: Cells were solubilized in 1% NP-40 buffer and the total protein concentration of the lysate was determined using the Bio-Rad protein assay.

    Techniques: Infection, Incubation, FLAG-tag, SDS Page, Western Blot, Purification, Migration

    Fusion loop gB mutants complex with gH/gL. ARPE-19 cells were transduced with Ad vectors expressing wild type gB (WT gB) or mutant forms of gB: gBA154W, gBW240A, or gBΔCT alone or in combination with gH/gL. Extracts were made using 1% NP-40 and gH/gL IP’d from extracts using MAb 14-4b. Western blots were probed with anti-gB rabbit polyclonal serum.

    Journal: PLoS Pathogens

    Article Title: Human Cytomegalovirus gH/gL Forms a Stable Complex with the Fusion Protein gB in Virions

    doi: 10.1371/journal.ppat.1005564

    Figure Lengend Snippet: Fusion loop gB mutants complex with gH/gL. ARPE-19 cells were transduced with Ad vectors expressing wild type gB (WT gB) or mutant forms of gB: gBA154W, gBW240A, or gBΔCT alone or in combination with gH/gL. Extracts were made using 1% NP-40 and gH/gL IP’d from extracts using MAb 14-4b. Western blots were probed with anti-gB rabbit polyclonal serum.

    Article Snippet: Cells were solubilized in 1% NP-40 buffer and the total protein concentration of the lysate was determined using the Bio-Rad protein assay.

    Techniques: Transduction, Expressing, Mutagenesis, Western Blot

    Kinetics of [ 3 H]cholesterol binding to purified NPC proteins. ( A  and  B ) Time course at different temperatures. Each reaction, in a final volume of 80 μl of buffer B (pH 6.5) with 0.004% Nonidet P-40, contained 4 pmol of NPC1(NTD)-LVPRGS-His-8-FLAG

    Journal:

    Article Title: NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes

    doi: 10.1073/pnas.0807328105

    Figure Lengend Snippet: Kinetics of [ 3 H]cholesterol binding to purified NPC proteins. ( A and B ) Time course at different temperatures. Each reaction, in a final volume of 80 μl of buffer B (pH 6.5) with 0.004% Nonidet P-40, contained 4 pmol of NPC1(NTD)-LVPRGS-His-8-FLAG

    Article Snippet: After incubation for 30 min at 4°C or 37°C, each reaction mixture was diluted with 500 μl of buffer A supplemented with 0.004% Nonidet P-40 and loaded onto a 2-ml column (Bio-Rad) packed with 0.3 ml of Ni-NTA-agarose beads (Qiagen) that had been preequilibrated with buffer A with 0.004% Nonidet P-40.

    Techniques: Binding Assay, Purification

    Segregation of Msp and CTLP in Triton X-114 extracts, as shown by Western immunoblot analysis. Samples were not heated before electrophoresis. The blots were probed with a mixture of antisera raised against Msp and CTLP. Lanes: 1, crude Triton X-114 extract; 2, extract following high-speed centrifugation; 3, aqueous phase of Triton X-114 extract; 4, detergent phase of Triton X-114 extract. Arrows: Msp oligomers with an apparent molecular mass of approximately 150 kDa; CTLP doublet at approximately 95 kDa.

    Journal: Infection and Immunity

    Article Title: Cytopathic Effects of the Major Surface Protein and the Chymotrypsinlike Protease of Treponema denticola

    doi:

    Figure Lengend Snippet: Segregation of Msp and CTLP in Triton X-114 extracts, as shown by Western immunoblot analysis. Samples were not heated before electrophoresis. The blots were probed with a mixture of antisera raised against Msp and CTLP. Lanes: 1, crude Triton X-114 extract; 2, extract following high-speed centrifugation; 3, aqueous phase of Triton X-114 extract; 4, detergent phase of Triton X-114 extract. Arrows: Msp oligomers with an apparent molecular mass of approximately 150 kDa; CTLP doublet at approximately 95 kDa.

    Article Snippet: Msp and CTLP were purified from the aqueous and detergent phases, respectively, of Triton X-114 extracts of 3-liter batch cultures of T. denticola by preparative SDS-PAGE with a model 491 Prep Cell (Bio-Rad Laboratories, Richmond, Calif.).

    Techniques: Western Blot, Electrophoresis, Centrifugation

    MSP2(P44) MAb 20B4-screened Western blot of  A. phagocytophilum  HGE1 whole-cell lysate and Triton X-114-partitioned hydrophobic and hydrophilic liquid fractions. Ten micrograms each of  A. phagocytophilum  HGE1 whole-cell lysate and the hydrophobic liquid

    Journal: Infection and Immunity

    Article Title: Anaplasma phagocytophilum MSP2(P44)-18 Predominates and Is Modified into Multiple Isoforms in Human Myeloid Cells

    doi: 10.1128/IAI.01594-07

    Figure Lengend Snippet: MSP2(P44) MAb 20B4-screened Western blot of A. phagocytophilum HGE1 whole-cell lysate and Triton X-114-partitioned hydrophobic and hydrophilic liquid fractions. Ten micrograms each of A. phagocytophilum HGE1 whole-cell lysate and the hydrophobic liquid

    Article Snippet: Bacterial outer membrane fractions were enriched for by temperature-dependent Triton X-114 phase partitioning ( , ) using the Membrane I ReadyPrep protein extraction kit (Bio-Rad, Hercules, CA) with minor modifications to the manufacturer's protocol.

    Techniques: Western Blot