np 40 substitute  (Millipore)


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    Nonidet P 40 Substitute solution
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    98379
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    Millipore np 40 substitute
    Nonidet P 40 Substitute solution

    https://www.bioz.com/result/np 40 substitute/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    np 40 substitute - by Bioz Stars, 2020-11
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    1) Product Images from "Lack of specificity of antibodies raised against CLN3, the lysosomal/endosomal transmembrane protein mutated in juvenile Batten disease"

    Article Title: Lack of specificity of antibodies raised against CLN3, the lysosomal/endosomal transmembrane protein mutated in juvenile Batten disease

    Journal: Bioscience Reports

    doi: 10.1042/BSR20171229

    Anti-CLN3 antibodies detect the same protein bands in wild-type (WT) and Cln3 −/− mouse brain extracts Three different anti-CLN3 antibodies [Abnova rabbit anti-CLN3 (D01P), Abnova mouse anti-CLN3 (M03), and Abcam rabbit anti-CLN3 (ab75959)] were tested in immunoblot experiments using protein extracts of surface cross-linked cerebellum and cortex tissue samples from 1-month-old WT and Cln3 −/− male mice. Protein extracts were prepared by sonication in a lysis buffer containing 500 mM NaCl, 0.1% NP-40 substitute and protease, and phosphatase inhibitor cocktails. Sixty micrograms of protein were loaded in each lane of SDS-containing 10% polyacrylamide gels. Prior to loading, samples were treated as indicated in reducing sample buffer without or with 4 M urea. After the electrophoretic separation, proteins were transferred onto nitrocellulose membranes and probed with the anti-CLN3 antibodies (Abnova rabbit, 1:500; Abnova mouse, 1:500; Abcam rabbit, 1:700); M.W. marker: PageRuler Plus (Thermo Fisher Scientific). The immunoblots shown are representative of two separate experiments.
    Figure Legend Snippet: Anti-CLN3 antibodies detect the same protein bands in wild-type (WT) and Cln3 −/− mouse brain extracts Three different anti-CLN3 antibodies [Abnova rabbit anti-CLN3 (D01P), Abnova mouse anti-CLN3 (M03), and Abcam rabbit anti-CLN3 (ab75959)] were tested in immunoblot experiments using protein extracts of surface cross-linked cerebellum and cortex tissue samples from 1-month-old WT and Cln3 −/− male mice. Protein extracts were prepared by sonication in a lysis buffer containing 500 mM NaCl, 0.1% NP-40 substitute and protease, and phosphatase inhibitor cocktails. Sixty micrograms of protein were loaded in each lane of SDS-containing 10% polyacrylamide gels. Prior to loading, samples were treated as indicated in reducing sample buffer without or with 4 M urea. After the electrophoretic separation, proteins were transferred onto nitrocellulose membranes and probed with the anti-CLN3 antibodies (Abnova rabbit, 1:500; Abnova mouse, 1:500; Abcam rabbit, 1:700); M.W. marker: PageRuler Plus (Thermo Fisher Scientific). The immunoblots shown are representative of two separate experiments.

    Techniques Used: Mouse Assay, Sonication, Lysis, Marker, Western Blot

    Related Articles

    Centrifugation:

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    Article Snippet: .. After centrifugation, the supernatant (400 μl) was incubated with 3 μl of rabbit antiserum for SSBP1 (αmSSBP1-1) at 4 °C for 1 h, and mixed with 40 μl protein A- or protein G-Sepharose beads (GE Healthcare) by rotating at 4 °C for 1 h. The complexes were washed with NP-40 lysis buffer, and were subjected to western blotting using mouse monoclonal IgG for Flag peptide (M2, Sigma-Aldrich). .. Immunofluorescence HeLa cells cultured on glass coverslips in 6 cm dishes at 37 °C for 24 h were infected for 2 h with adenovirus expressing scrambled RNA or short hairpin RNA for HSF1 or each VDAC protein (1 × 107 p.f.u. per ml), and then maintained with normal medium for 70 h. After being heat-shocked at 42 °C for 60 min, the cells were fixed with 4% paraformaldehyde in medium at room temperature for 10 min.

    Article Title: Dynamic Interactions of the UL16 Tegument Protein with the Capsid of Herpes Simplex Virus ▿
    Article Snippet: .. At 16 to 20 h postinfection, cells were scraped into 20 ml of PBS, collected by centrifugation at 1,000 × g for 10 min, resuspended in 6 ml of NP-40 lysis buffer (0.5% NP-40, 150 mM NaCl, 1 M Tris-HCl, pH 8.0) containing protease inhibitors (P8340; Sigma), and incubated for 15 min on ice. .. The cytoplasmic fraction was separated from the nuclei by centrifugation at 1,000 × g for 10 min.

    Magnetic Beads:

    Article Title: Structural basis of thalidomide enantiomer binding to cereblon
    Article Snippet: .. Extracts were incubated with M2 FLAG magnetic beads (Sigma) in the presence of ( S )-D-thalidomide or ( R )-D-thalidomide and incubated for 2 h. Following extensive washing three times with 0.5% NP-40 lysis buffer, bound proteins were eluted with free 3x FLAG-peptides (Sigma) and then subjected to immunoblot analysis. .. Binding studies utilizing isothermal titration calorimetry (ITC) were conducted using a calorimeter (MicroCal iTC200 , GE Healthcare) at 20 °C.

    Mutagenesis:

    Article Title: Interaction and Interdependent Packaging of Tegument Protein UL11 and Glycoprotein E of Herpes Simplex Virus ▿
    Article Snippet: .. At 18 to 24 h posttransfection, the cells were harvested in NP-40 lysis buffer (0.5% NP-40, 150 mM NaCl, 50 mM Tris-HCl [pH 8.0], protease inhibitor cocktail [P8340; Sigma]), precleared with glutathione-Sepharose 4B beads for 2 h at room temperature, and then incubated for 5 h at room temperature with wild-type or mutant GST-gE.CT proteins immobilized on glutathione-Sepharose beads. .. The beads were washed three times with NP-40 buffer for 10 min each and boiled for 5 min, and the proteins bound to the beads were separated by SDS-PAGE, transferred to nitrocellulose membranes, probed with proper antibodies, and developed by ELC Western blotting system (Pierce).

    Protease Inhibitor:

    Article Title: Lack of specificity of antibodies raised against CLN3, the lysosomal/endosomal transmembrane protein mutated in juvenile Batten disease
    Article Snippet: .. Briefly, the surface cross-linked tissue slices in 1.5-ml microtubes were homogenized by sonication in ice-cold lysis buffer [25 mM HEPES (pH 7.4), 500 mM NaCl, 2 mM EDTA, 20 mM NaF, 1 mM sodium orthovanadate, 0.1% NP-40 substitute, 1 mM DTT, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Sigma)]. ..

    Article Title: Interaction and Interdependent Packaging of Tegument Protein UL11 and Glycoprotein E of Herpes Simplex Virus ▿
    Article Snippet: .. At 18 to 24 h posttransfection, the cells were harvested in NP-40 lysis buffer (0.5% NP-40, 150 mM NaCl, 50 mM Tris-HCl [pH 8.0], protease inhibitor cocktail [P8340; Sigma]), precleared with glutathione-Sepharose 4B beads for 2 h at room temperature, and then incubated for 5 h at room temperature with wild-type or mutant GST-gE.CT proteins immobilized on glutathione-Sepharose beads. .. The beads were washed three times with NP-40 buffer for 10 min each and boiled for 5 min, and the proteins bound to the beads were separated by SDS-PAGE, transferred to nitrocellulose membranes, probed with proper antibodies, and developed by ELC Western blotting system (Pierce).

    Article Title: Glutaredoxin Regulates Apoptosis in Cardiomyocytes via NF?B Targets Bcl-2 and Bcl-xL: Implications for Cardiac Aging
    Article Snippet: .. H9c2 cells were lysed in NP-40 lysis buffer containing Sigma Protease Inhibitor Cocktail (5%, final). .. The cytosolic fraction of F344 rat heart homogenate was prepared as described above for analysis of Grx activity.

    Incubation:

    Article Title: Structural basis of thalidomide enantiomer binding to cereblon
    Article Snippet: .. Extracts were incubated with M2 FLAG magnetic beads (Sigma) in the presence of ( S )-D-thalidomide or ( R )-D-thalidomide and incubated for 2 h. Following extensive washing three times with 0.5% NP-40 lysis buffer, bound proteins were eluted with free 3x FLAG-peptides (Sigma) and then subjected to immunoblot analysis. .. Binding studies utilizing isothermal titration calorimetry (ITC) were conducted using a calorimeter (MicroCal iTC200 , GE Healthcare) at 20 °C.

    Article Title: Mitochondrial SSBP1 protects cells from proteotoxic stresses by potentiating stress-induced HSF1 transcriptional activity
    Article Snippet: .. After centrifugation, the supernatant (400 μl) was incubated with 3 μl of rabbit antiserum for SSBP1 (αmSSBP1-1) at 4 °C for 1 h, and mixed with 40 μl protein A- or protein G-Sepharose beads (GE Healthcare) by rotating at 4 °C for 1 h. The complexes were washed with NP-40 lysis buffer, and were subjected to western blotting using mouse monoclonal IgG for Flag peptide (M2, Sigma-Aldrich). .. Immunofluorescence HeLa cells cultured on glass coverslips in 6 cm dishes at 37 °C for 24 h were infected for 2 h with adenovirus expressing scrambled RNA or short hairpin RNA for HSF1 or each VDAC protein (1 × 107 p.f.u. per ml), and then maintained with normal medium for 70 h. After being heat-shocked at 42 °C for 60 min, the cells were fixed with 4% paraformaldehyde in medium at room temperature for 10 min.

    Article Title: Interaction and Interdependent Packaging of Tegument Protein UL11 and Glycoprotein E of Herpes Simplex Virus ▿
    Article Snippet: .. At 18 to 24 h posttransfection, the cells were harvested in NP-40 lysis buffer (0.5% NP-40, 150 mM NaCl, 50 mM Tris-HCl [pH 8.0], protease inhibitor cocktail [P8340; Sigma]), precleared with glutathione-Sepharose 4B beads for 2 h at room temperature, and then incubated for 5 h at room temperature with wild-type or mutant GST-gE.CT proteins immobilized on glutathione-Sepharose beads. .. The beads were washed three times with NP-40 buffer for 10 min each and boiled for 5 min, and the proteins bound to the beads were separated by SDS-PAGE, transferred to nitrocellulose membranes, probed with proper antibodies, and developed by ELC Western blotting system (Pierce).

    Article Title: Dynamic Interactions of the UL16 Tegument Protein with the Capsid of Herpes Simplex Virus ▿
    Article Snippet: .. At 16 to 20 h postinfection, cells were scraped into 20 ml of PBS, collected by centrifugation at 1,000 × g for 10 min, resuspended in 6 ml of NP-40 lysis buffer (0.5% NP-40, 150 mM NaCl, 1 M Tris-HCl, pH 8.0) containing protease inhibitors (P8340; Sigma), and incubated for 15 min on ice. .. The cytoplasmic fraction was separated from the nuclei by centrifugation at 1,000 × g for 10 min.

    Sonication:

    Article Title: Lack of specificity of antibodies raised against CLN3, the lysosomal/endosomal transmembrane protein mutated in juvenile Batten disease
    Article Snippet: .. Briefly, the surface cross-linked tissue slices in 1.5-ml microtubes were homogenized by sonication in ice-cold lysis buffer [25 mM HEPES (pH 7.4), 500 mM NaCl, 2 mM EDTA, 20 mM NaF, 1 mM sodium orthovanadate, 0.1% NP-40 substitute, 1 mM DTT, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Sigma)]. ..

    Western Blot:

    Article Title: Mitochondrial SSBP1 protects cells from proteotoxic stresses by potentiating stress-induced HSF1 transcriptional activity
    Article Snippet: .. After centrifugation, the supernatant (400 μl) was incubated with 3 μl of rabbit antiserum for SSBP1 (αmSSBP1-1) at 4 °C for 1 h, and mixed with 40 μl protein A- or protein G-Sepharose beads (GE Healthcare) by rotating at 4 °C for 1 h. The complexes were washed with NP-40 lysis buffer, and were subjected to western blotting using mouse monoclonal IgG for Flag peptide (M2, Sigma-Aldrich). .. Immunofluorescence HeLa cells cultured on glass coverslips in 6 cm dishes at 37 °C for 24 h were infected for 2 h with adenovirus expressing scrambled RNA or short hairpin RNA for HSF1 or each VDAC protein (1 × 107 p.f.u. per ml), and then maintained with normal medium for 70 h. After being heat-shocked at 42 °C for 60 min, the cells were fixed with 4% paraformaldehyde in medium at room temperature for 10 min.

    Lysis:

    Article Title: Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿Epstein-Barr Virus LF2 Protein Regulates Viral Replication by Altering Rta Subcellular Localization ▿ †
    Article Snippet: .. The beads were washed extensively with NP-40 lysis buffer (1% [vol/vol] Igepal CA-630, 20 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 5 mM MgCl2 ) and eluted with 0.4 mg/ml Flag peptide (Sigma) in NP-40 lysis buffer. ..

    Article Title: Lack of specificity of antibodies raised against CLN3, the lysosomal/endosomal transmembrane protein mutated in juvenile Batten disease
    Article Snippet: .. Briefly, the surface cross-linked tissue slices in 1.5-ml microtubes were homogenized by sonication in ice-cold lysis buffer [25 mM HEPES (pH 7.4), 500 mM NaCl, 2 mM EDTA, 20 mM NaF, 1 mM sodium orthovanadate, 0.1% NP-40 substitute, 1 mM DTT, protease inhibitor cocktail, and phosphatase inhibitor cocktail (Sigma)]. ..

    Article Title: Structural basis of thalidomide enantiomer binding to cereblon
    Article Snippet: .. Extracts were incubated with M2 FLAG magnetic beads (Sigma) in the presence of ( S )-D-thalidomide or ( R )-D-thalidomide and incubated for 2 h. Following extensive washing three times with 0.5% NP-40 lysis buffer, bound proteins were eluted with free 3x FLAG-peptides (Sigma) and then subjected to immunoblot analysis. .. Binding studies utilizing isothermal titration calorimetry (ITC) were conducted using a calorimeter (MicroCal iTC200 , GE Healthcare) at 20 °C.

    Article Title: Cellular and Molecular Requirements for Association of the Murine Cytomegalovirus Protein m4/gp34 with Major Histocompatibility Complex Class I Molecules
    Article Snippet: .. Cells were lysed in NP-40 lysis buffer (0.5% NP-40, 50 mM Tris-HCl, pH 6.5, 5 mM MgCl2 ) or digitonin lysis buffer (1% high-purity digitonin [Calbiochem, La Jolla, CA] in freshly prepared phosphate-buffered saline, pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride. .. Nuclei and insoluble debris were removed by centrifugation (Savant SFA13K centrifuge; 13,000 rpm, 10 min).

    Article Title: Mitochondrial SSBP1 protects cells from proteotoxic stresses by potentiating stress-induced HSF1 transcriptional activity
    Article Snippet: .. After centrifugation, the supernatant (400 μl) was incubated with 3 μl of rabbit antiserum for SSBP1 (αmSSBP1-1) at 4 °C for 1 h, and mixed with 40 μl protein A- or protein G-Sepharose beads (GE Healthcare) by rotating at 4 °C for 1 h. The complexes were washed with NP-40 lysis buffer, and were subjected to western blotting using mouse monoclonal IgG for Flag peptide (M2, Sigma-Aldrich). .. Immunofluorescence HeLa cells cultured on glass coverslips in 6 cm dishes at 37 °C for 24 h were infected for 2 h with adenovirus expressing scrambled RNA or short hairpin RNA for HSF1 or each VDAC protein (1 × 107 p.f.u. per ml), and then maintained with normal medium for 70 h. After being heat-shocked at 42 °C for 60 min, the cells were fixed with 4% paraformaldehyde in medium at room temperature for 10 min.

    Article Title: Dynamic Interactions of the UL16 Tegument Protein with the Capsid of Herpes Simplex Virus ▿
    Article Snippet: .. At 16 to 20 h postinfection, cells were scraped into 20 ml of PBS, collected by centrifugation at 1,000 × g for 10 min, resuspended in 6 ml of NP-40 lysis buffer (0.5% NP-40, 150 mM NaCl, 1 M Tris-HCl, pH 8.0) containing protease inhibitors (P8340; Sigma), and incubated for 15 min on ice. .. The cytoplasmic fraction was separated from the nuclei by centrifugation at 1,000 × g for 10 min.

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    Millipore np 40 lysis buffer
    Role of the peptide-loading complex in m4/gp34-K b association. (A) Effects of peptide-loading complex on the association of m4/gp34 with K b molecules in the context of MCMV infection. TAP −/− , tapasin −/− (Tpn −/− ), or β 2 m −/− fibroblasts or normal MEFs from C57BL/6 mice were infected with wild-type MCMV (MOI = 20) and labeled overnight with 35 S. The cells were lysed in <t>NP-40</t> lysis buffer and precleared with normal rabbit serum, followed by sequential IP with the polyclonal antiserum anti-p8 to precipitate K b and its associated molecules and with antiserum 8139 to precipitate non-K b -associated free m4/gp34. (B) m4/gp34-K b complexes associate with tapasin. IFN-γ-treated B6 MEFs or K b D b−/− fibroblasts were infected with either wild-type or Δm4 MCMV for 3.5 h and then labeled with 35 S for 2 h. The cells were lysed in digitonin lysis buffer. Tapasin was immunoprecipitated from the precleared lysates. A control IP was performed with normal rabbit serum (NRS). The immune complexes were dissociated by boiling in 1% SDS for 10 min, resuspended in 1% NP-40 lysis buffer, and subjected to secondary IP against m4/gp34. Samples were treated with endo-H and analyzed by SDS-PAGE.
    Np 40 Lysis Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 529 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/np 40 lysis buffer/product/Millipore
    Average 99 stars, based on 529 article reviews
    Price from $9.99 to $1999.99
    np 40 lysis buffer - by Bioz Stars, 2020-11
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    Role of the peptide-loading complex in m4/gp34-K b association. (A) Effects of peptide-loading complex on the association of m4/gp34 with K b molecules in the context of MCMV infection. TAP −/− , tapasin −/− (Tpn −/− ), or β 2 m −/− fibroblasts or normal MEFs from C57BL/6 mice were infected with wild-type MCMV (MOI = 20) and labeled overnight with 35 S. The cells were lysed in NP-40 lysis buffer and precleared with normal rabbit serum, followed by sequential IP with the polyclonal antiserum anti-p8 to precipitate K b and its associated molecules and with antiserum 8139 to precipitate non-K b -associated free m4/gp34. (B) m4/gp34-K b complexes associate with tapasin. IFN-γ-treated B6 MEFs or K b D b−/− fibroblasts were infected with either wild-type or Δm4 MCMV for 3.5 h and then labeled with 35 S for 2 h. The cells were lysed in digitonin lysis buffer. Tapasin was immunoprecipitated from the precleared lysates. A control IP was performed with normal rabbit serum (NRS). The immune complexes were dissociated by boiling in 1% SDS for 10 min, resuspended in 1% NP-40 lysis buffer, and subjected to secondary IP against m4/gp34. Samples were treated with endo-H and analyzed by SDS-PAGE.

    Journal: Journal of Virology

    Article Title: Cellular and Molecular Requirements for Association of the Murine Cytomegalovirus Protein m4/gp34 with Major Histocompatibility Complex Class I Molecules

    doi: 10.1128/JVI.00534-06

    Figure Lengend Snippet: Role of the peptide-loading complex in m4/gp34-K b association. (A) Effects of peptide-loading complex on the association of m4/gp34 with K b molecules in the context of MCMV infection. TAP −/− , tapasin −/− (Tpn −/− ), or β 2 m −/− fibroblasts or normal MEFs from C57BL/6 mice were infected with wild-type MCMV (MOI = 20) and labeled overnight with 35 S. The cells were lysed in NP-40 lysis buffer and precleared with normal rabbit serum, followed by sequential IP with the polyclonal antiserum anti-p8 to precipitate K b and its associated molecules and with antiserum 8139 to precipitate non-K b -associated free m4/gp34. (B) m4/gp34-K b complexes associate with tapasin. IFN-γ-treated B6 MEFs or K b D b−/− fibroblasts were infected with either wild-type or Δm4 MCMV for 3.5 h and then labeled with 35 S for 2 h. The cells were lysed in digitonin lysis buffer. Tapasin was immunoprecipitated from the precleared lysates. A control IP was performed with normal rabbit serum (NRS). The immune complexes were dissociated by boiling in 1% SDS for 10 min, resuspended in 1% NP-40 lysis buffer, and subjected to secondary IP against m4/gp34. Samples were treated with endo-H and analyzed by SDS-PAGE.

    Article Snippet: Cells were lysed in NP-40 lysis buffer (0.5% NP-40, 50 mM Tris-HCl, pH 6.5, 5 mM MgCl2 ) or digitonin lysis buffer (1% high-purity digitonin [Calbiochem, La Jolla, CA] in freshly prepared phosphate-buffered saline, pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride.

    Techniques: Infection, Mouse Assay, Labeling, Lysis, Immunoprecipitation, SDS Page

    Association of m4/gp34 with K b in detergent lysates. K b D b−/− cells were infected with Ad-m4 for 4 h, followed by overnight labeling with 35 S. TAP −/− cells were labeled overnight without infection. The next morning, the cells were lysed in NP-40 lysis buffer supplemented with 1 mM phenylmethylsulfonyl fluoride. After the lysates were precleared with normal rabbit serum, equal volumes of TAP −/− lysate and Ad-m4-infected K b D b−/− lysate (K b D b−/− /Ad-m4) were mixed and incubated overnight at 4°C with the indicated concentration of the SIINFEKL peptide. The mixed lysates were then sequentially immunoprecipitated with the antibody anti-p8 and the m4/gp34-specific antiserum 8139. All samples were treated with endo-H.

    Journal: Journal of Virology

    Article Title: Cellular and Molecular Requirements for Association of the Murine Cytomegalovirus Protein m4/gp34 with Major Histocompatibility Complex Class I Molecules

    doi: 10.1128/JVI.00534-06

    Figure Lengend Snippet: Association of m4/gp34 with K b in detergent lysates. K b D b−/− cells were infected with Ad-m4 for 4 h, followed by overnight labeling with 35 S. TAP −/− cells were labeled overnight without infection. The next morning, the cells were lysed in NP-40 lysis buffer supplemented with 1 mM phenylmethylsulfonyl fluoride. After the lysates were precleared with normal rabbit serum, equal volumes of TAP −/− lysate and Ad-m4-infected K b D b−/− lysate (K b D b−/− /Ad-m4) were mixed and incubated overnight at 4°C with the indicated concentration of the SIINFEKL peptide. The mixed lysates were then sequentially immunoprecipitated with the antibody anti-p8 and the m4/gp34-specific antiserum 8139. All samples were treated with endo-H.

    Article Snippet: Cells were lysed in NP-40 lysis buffer (0.5% NP-40, 50 mM Tris-HCl, pH 6.5, 5 mM MgCl2 ) or digitonin lysis buffer (1% high-purity digitonin [Calbiochem, La Jolla, CA] in freshly prepared phosphate-buffered saline, pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride.

    Techniques: Infection, Labeling, Lysis, Incubation, Concentration Assay, Immunoprecipitation