nitrocellulose membrane Search Results


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    Nitrocellulose membranes are a popular matrix used in protein blotting because of their high protein binding affinity compatibility with a variety of detection methods chemiluminescence chromogenic and fluorescence and the
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    99
    LI-COR nitrocellulose membrane
    Nitrocellulose Membrane, supplied by LI-COR, used in various techniques. Bioz Stars score: 99/100, based on 2119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad nitrocellulose membranes
    Nitrocellulose Membranes, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 25446 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Millipore nitrocellulose membranes
    Nitrocellulose Membranes, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 11816 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher nitrocellulose membrane
    Nitrocellulose Membrane, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18517 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Schuell GmbH nitrocellulose membrane
    Nitrocellulose Membrane, supplied by Schuell GmbH, used in various techniques. Bioz Stars score: 92/100, based on 8304 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare nitrocellulose membrane
    Nitrocellulose Membrane, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 43130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare hybond ecl nitrocellulose membranes
    Hybond Ecl Nitrocellulose Membranes, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 3042 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore membranes
    Membranes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 9063 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher membranes
    Membranes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5128 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam nitrocellulose membranes
    Nitrocellulose Membranes, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1678 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc nitrocellulose membrane
    Nitrocellulose Membrane, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 3326 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Pall Corporation nitrocellulose membranes
    Nitrocellulose Membranes, supplied by Pall Corporation, used in various techniques. Bioz Stars score: 92/100, based on 1074 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore nitrocellulose filters
    Nitrocellulose Filters, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1852 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck KGaA nitrocellulose membrane
    Nitrocellulose Membrane, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 958 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Schuell GmbH protran nitrocellulose membranes
    scFv-VSV-G expression plasmids and incorporation in HIV-1 derived particles. a) Expression plasmids: (I) VSV-G expression plasmid (PM 730). The 1.6 kB HindIII-BamHI VSV-G fragment (serotype Indiana) was transferred from pFB.VSVG (J.M. Heard, Paris, France) into pcDNA3 (InVitrogen) by PCR cloning according to standard procedures. A haemagglutinin (HA) sequence was added at the C terminus of VSV-G for immunodetection. (II and III) scFv/VSV-G expression plasmids. The chimaeric constructs were generated by PCR-based cloning. Mature VSV-G (from amino acid 17) was amplified from PM 730 and introduced into the PM 441 and PM 442 plasmids [23]. These constructs originate from an MLV-derived plasmid (FBMOSALF [31]), modified to contain a scFv (αMHC and αHEL, respectively [27]), upstream of the GP gene. Consequently, the resulting constructs (αMHC/VSV-G and αHEL/VSV-G) express the genes from the MLV LTR, with a MLV leader sequence (L mlv ) and 6 additional amino acids from the virus. (IV and V) Vectors for production of HIV-1 derived viral particles. A HIV-1-based lentiviral vector (V) (CD 416; pHRCMV) [29], into which EGFP gene had been inserted, together with a helper plasmid (CD 417; pCMV△R8.2) [29] expressing Gag, Pol and accessory HIV-1 proteins (IV), were used for production of HIV-1 particles. Expression vectors, physical maps and primer sequences are available upon request. b. VSV-G- and scFv/VSV-G pseudotyped HIV-1 particles, produced in 293T cells. 2 × 10 6 293T cells in 10 cm diameter tissue culture dishes were transiently transfected with 5 μg of an LTR-driven EGFP vector (pHRCMV-EGFP) and 4 μg of a helper plasmid (pCMV△R8.2) [29], using the calcium phosphate co-precipitation procedure [36]. 5 μg PM 730 or 30 μg scFv/VSV-G plasmid (PM 981 or PM 983) were also included. DNA precipitates were removed after 16 hours, and the viral supernatants were collected 24–48 hours later and pelleted by ultracentrifugation (BeckmanCoulter) at 25 kRPM, 4°C for 2 hours and resuspended in 1% of the original volume. Cell lysates and 2 μl concentrated scFv/VSV-G virus or 10 μl of non-concentrated VSV-G virus were separated on a 12 % SDS polyacrylamide gel and transferred onto <t>Protran</t> nitrocellulose membranes (Schleicher and Schuell). HA-tagged VSV-G and scFv/VSV-G were detected using a rat anti-HA antibody (Sigma), followed by horse radish peroxidase (HPO) conjugated anti-RatIgG (Dako). p24Gag was detected using SF2 rabbit monoclonal antibody (NIH AIDS Research and Reference Reagent Program) followed by anti-rabbit IgG/HPO (Santa Cruz), and was used as an internal reference to normalise for the virion protein quantities. The membranes were developed with Renaissance chemoluminescence kit (NEN Life Science Products), as recommended by the supplier.
    Protran Nitrocellulose Membranes, supplied by Schuell GmbH, used in various techniques. Bioz Stars score: 92/100, based on 652 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    PerkinElmer nitrocellulose membrane
    scFv-VSV-G expression plasmids and incorporation in HIV-1 derived particles. a) Expression plasmids: (I) VSV-G expression plasmid (PM 730). The 1.6 kB HindIII-BamHI VSV-G fragment (serotype Indiana) was transferred from pFB.VSVG (J.M. Heard, Paris, France) into pcDNA3 (InVitrogen) by PCR cloning according to standard procedures. A haemagglutinin (HA) sequence was added at the C terminus of VSV-G for immunodetection. (II and III) scFv/VSV-G expression plasmids. The chimaeric constructs were generated by PCR-based cloning. Mature VSV-G (from amino acid 17) was amplified from PM 730 and introduced into the PM 441 and PM 442 plasmids [23]. These constructs originate from an MLV-derived plasmid (FBMOSALF [31]), modified to contain a scFv (αMHC and αHEL, respectively [27]), upstream of the GP gene. Consequently, the resulting constructs (αMHC/VSV-G and αHEL/VSV-G) express the genes from the MLV LTR, with a MLV leader sequence (L mlv ) and 6 additional amino acids from the virus. (IV and V) Vectors for production of HIV-1 derived viral particles. A HIV-1-based lentiviral vector (V) (CD 416; pHRCMV) [29], into which EGFP gene had been inserted, together with a helper plasmid (CD 417; pCMV△R8.2) [29] expressing Gag, Pol and accessory HIV-1 proteins (IV), were used for production of HIV-1 particles. Expression vectors, physical maps and primer sequences are available upon request. b. VSV-G- and scFv/VSV-G pseudotyped HIV-1 particles, produced in 293T cells. 2 × 10 6 293T cells in 10 cm diameter tissue culture dishes were transiently transfected with 5 μg of an LTR-driven EGFP vector (pHRCMV-EGFP) and 4 μg of a helper plasmid (pCMV△R8.2) [29], using the calcium phosphate co-precipitation procedure [36]. 5 μg PM 730 or 30 μg scFv/VSV-G plasmid (PM 981 or PM 983) were also included. DNA precipitates were removed after 16 hours, and the viral supernatants were collected 24–48 hours later and pelleted by ultracentrifugation (BeckmanCoulter) at 25 kRPM, 4°C for 2 hours and resuspended in 1% of the original volume. Cell lysates and 2 μl concentrated scFv/VSV-G virus or 10 μl of non-concentrated VSV-G virus were separated on a 12 % SDS polyacrylamide gel and transferred onto <t>Protran</t> nitrocellulose membranes (Schleicher and Schuell). HA-tagged VSV-G and scFv/VSV-G were detected using a rat anti-HA antibody (Sigma), followed by horse radish peroxidase (HPO) conjugated anti-RatIgG (Dako). p24Gag was detected using SF2 rabbit monoclonal antibody (NIH AIDS Research and Reference Reagent Program) followed by anti-rabbit IgG/HPO (Santa Cruz), and was used as an internal reference to normalise for the virion protein quantities. The membranes were developed with Renaissance chemoluminescence kit (NEN Life Science Products), as recommended by the supplier.
    Nitrocellulose Membrane, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 93/100, based on 582 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Bio-Rad trans blot nitrocellulose membranes
    scFv-VSV-G expression plasmids and incorporation in HIV-1 derived particles. a) Expression plasmids: (I) VSV-G expression plasmid (PM 730). The 1.6 kB HindIII-BamHI VSV-G fragment (serotype Indiana) was transferred from pFB.VSVG (J.M. Heard, Paris, France) into pcDNA3 (InVitrogen) by PCR cloning according to standard procedures. A haemagglutinin (HA) sequence was added at the C terminus of VSV-G for immunodetection. (II and III) scFv/VSV-G expression plasmids. The chimaeric constructs were generated by PCR-based cloning. Mature VSV-G (from amino acid 17) was amplified from PM 730 and introduced into the PM 441 and PM 442 plasmids [23]. These constructs originate from an MLV-derived plasmid (FBMOSALF [31]), modified to contain a scFv (αMHC and αHEL, respectively [27]), upstream of the GP gene. Consequently, the resulting constructs (αMHC/VSV-G and αHEL/VSV-G) express the genes from the MLV LTR, with a MLV leader sequence (L mlv ) and 6 additional amino acids from the virus. (IV and V) Vectors for production of HIV-1 derived viral particles. A HIV-1-based lentiviral vector (V) (CD 416; pHRCMV) [29], into which EGFP gene had been inserted, together with a helper plasmid (CD 417; pCMV△R8.2) [29] expressing Gag, Pol and accessory HIV-1 proteins (IV), were used for production of HIV-1 particles. Expression vectors, physical maps and primer sequences are available upon request. b. VSV-G- and scFv/VSV-G pseudotyped HIV-1 particles, produced in 293T cells. 2 × 10 6 293T cells in 10 cm diameter tissue culture dishes were transiently transfected with 5 μg of an LTR-driven EGFP vector (pHRCMV-EGFP) and 4 μg of a helper plasmid (pCMV△R8.2) [29], using the calcium phosphate co-precipitation procedure [36]. 5 μg PM 730 or 30 μg scFv/VSV-G plasmid (PM 981 or PM 983) were also included. DNA precipitates were removed after 16 hours, and the viral supernatants were collected 24–48 hours later and pelleted by ultracentrifugation (BeckmanCoulter) at 25 kRPM, 4°C for 2 hours and resuspended in 1% of the original volume. Cell lysates and 2 μl concentrated scFv/VSV-G virus or 10 μl of non-concentrated VSV-G virus were separated on a 12 % SDS polyacrylamide gel and transferred onto <t>Protran</t> nitrocellulose membranes (Schleicher and Schuell). HA-tagged VSV-G and scFv/VSV-G were detected using a rat anti-HA antibody (Sigma), followed by horse radish peroxidase (HPO) conjugated anti-RatIgG (Dako). p24Gag was detected using SF2 rabbit monoclonal antibody (NIH AIDS Research and Reference Reagent Program) followed by anti-rabbit IgG/HPO (Santa Cruz), and was used as an internal reference to normalise for the virion protein quantities. The membranes were developed with Renaissance chemoluminescence kit (NEN Life Science Products), as recommended by the supplier.
    Trans Blot Nitrocellulose Membranes, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 89/100, based on 382 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 89 stars, based on 382 article reviews
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    99
    Bio-Rad membranes
    scFv-VSV-G expression plasmids and incorporation in HIV-1 derived particles. a) Expression plasmids: (I) VSV-G expression plasmid (PM 730). The 1.6 kB HindIII-BamHI VSV-G fragment (serotype Indiana) was transferred from pFB.VSVG (J.M. Heard, Paris, France) into pcDNA3 (InVitrogen) by PCR cloning according to standard procedures. A haemagglutinin (HA) sequence was added at the C terminus of VSV-G for immunodetection. (II and III) scFv/VSV-G expression plasmids. The chimaeric constructs were generated by PCR-based cloning. Mature VSV-G (from amino acid 17) was amplified from PM 730 and introduced into the PM 441 and PM 442 plasmids [23]. These constructs originate from an MLV-derived plasmid (FBMOSALF [31]), modified to contain a scFv (αMHC and αHEL, respectively [27]), upstream of the GP gene. Consequently, the resulting constructs (αMHC/VSV-G and αHEL/VSV-G) express the genes from the MLV LTR, with a MLV leader sequence (L mlv ) and 6 additional amino acids from the virus. (IV and V) Vectors for production of HIV-1 derived viral particles. A HIV-1-based lentiviral vector (V) (CD 416; pHRCMV) [29], into which EGFP gene had been inserted, together with a helper plasmid (CD 417; pCMV△R8.2) [29] expressing Gag, Pol and accessory HIV-1 proteins (IV), were used for production of HIV-1 particles. Expression vectors, physical maps and primer sequences are available upon request. b. VSV-G- and scFv/VSV-G pseudotyped HIV-1 particles, produced in 293T cells. 2 × 10 6 293T cells in 10 cm diameter tissue culture dishes were transiently transfected with 5 μg of an LTR-driven EGFP vector (pHRCMV-EGFP) and 4 μg of a helper plasmid (pCMV△R8.2) [29], using the calcium phosphate co-precipitation procedure [36]. 5 μg PM 730 or 30 μg scFv/VSV-G plasmid (PM 981 or PM 983) were also included. DNA precipitates were removed after 16 hours, and the viral supernatants were collected 24–48 hours later and pelleted by ultracentrifugation (BeckmanCoulter) at 25 kRPM, 4°C for 2 hours and resuspended in 1% of the original volume. Cell lysates and 2 μl concentrated scFv/VSV-G virus or 10 μl of non-concentrated VSV-G virus were separated on a 12 % SDS polyacrylamide gel and transferred onto <t>Protran</t> nitrocellulose membranes (Schleicher and Schuell). HA-tagged VSV-G and scFv/VSV-G were detected using a rat anti-HA antibody (Sigma), followed by horse radish peroxidase (HPO) conjugated anti-RatIgG (Dako). p24Gag was detected using SF2 rabbit monoclonal antibody (NIH AIDS Research and Reference Reagent Program) followed by anti-rabbit IgG/HPO (Santa Cruz), and was used as an internal reference to normalise for the virion protein quantities. The membranes were developed with Renaissance chemoluminescence kit (NEN Life Science Products), as recommended by the supplier.
    Membranes, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 15858 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Osmonics nitrocellulose membranes
    scFv-VSV-G expression plasmids and incorporation in HIV-1 derived particles. a) Expression plasmids: (I) VSV-G expression plasmid (PM 730). The 1.6 kB HindIII-BamHI VSV-G fragment (serotype Indiana) was transferred from pFB.VSVG (J.M. Heard, Paris, France) into pcDNA3 (InVitrogen) by PCR cloning according to standard procedures. A haemagglutinin (HA) sequence was added at the C terminus of VSV-G for immunodetection. (II and III) scFv/VSV-G expression plasmids. The chimaeric constructs were generated by PCR-based cloning. Mature VSV-G (from amino acid 17) was amplified from PM 730 and introduced into the PM 441 and PM 442 plasmids [23]. These constructs originate from an MLV-derived plasmid (FBMOSALF [31]), modified to contain a scFv (αMHC and αHEL, respectively [27]), upstream of the GP gene. Consequently, the resulting constructs (αMHC/VSV-G and αHEL/VSV-G) express the genes from the MLV LTR, with a MLV leader sequence (L mlv ) and 6 additional amino acids from the virus. (IV and V) Vectors for production of HIV-1 derived viral particles. A HIV-1-based lentiviral vector (V) (CD 416; pHRCMV) [29], into which EGFP gene had been inserted, together with a helper plasmid (CD 417; pCMV△R8.2) [29] expressing Gag, Pol and accessory HIV-1 proteins (IV), were used for production of HIV-1 particles. Expression vectors, physical maps and primer sequences are available upon request. b. VSV-G- and scFv/VSV-G pseudotyped HIV-1 particles, produced in 293T cells. 2 × 10 6 293T cells in 10 cm diameter tissue culture dishes were transiently transfected with 5 μg of an LTR-driven EGFP vector (pHRCMV-EGFP) and 4 μg of a helper plasmid (pCMV△R8.2) [29], using the calcium phosphate co-precipitation procedure [36]. 5 μg PM 730 or 30 μg scFv/VSV-G plasmid (PM 981 or PM 983) were also included. DNA precipitates were removed after 16 hours, and the viral supernatants were collected 24–48 hours later and pelleted by ultracentrifugation (BeckmanCoulter) at 25 kRPM, 4°C for 2 hours and resuspended in 1% of the original volume. Cell lysates and 2 μl concentrated scFv/VSV-G virus or 10 μl of non-concentrated VSV-G virus were separated on a 12 % SDS polyacrylamide gel and transferred onto <t>Protran</t> nitrocellulose membranes (Schleicher and Schuell). HA-tagged VSV-G and scFv/VSV-G were detected using a rat anti-HA antibody (Sigma), followed by horse radish peroxidase (HPO) conjugated anti-RatIgG (Dako). p24Gag was detected using SF2 rabbit monoclonal antibody (NIH AIDS Research and Reference Reagent Program) followed by anti-rabbit IgG/HPO (Santa Cruz), and was used as an internal reference to normalise for the virion protein quantities. The membranes were developed with Renaissance chemoluminescence kit (NEN Life Science Products), as recommended by the supplier.
    Nitrocellulose Membranes, supplied by Osmonics, used in various techniques. Bioz Stars score: 92/100, based on 358 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Sartorius AG nitrocellulose membrane
    scFv-VSV-G expression plasmids and incorporation in HIV-1 derived particles. a) Expression plasmids: (I) VSV-G expression plasmid (PM 730). The 1.6 kB HindIII-BamHI VSV-G fragment (serotype Indiana) was transferred from pFB.VSVG (J.M. Heard, Paris, France) into pcDNA3 (InVitrogen) by PCR cloning according to standard procedures. A haemagglutinin (HA) sequence was added at the C terminus of VSV-G for immunodetection. (II and III) scFv/VSV-G expression plasmids. The chimaeric constructs were generated by PCR-based cloning. Mature VSV-G (from amino acid 17) was amplified from PM 730 and introduced into the PM 441 and PM 442 plasmids [23]. These constructs originate from an MLV-derived plasmid (FBMOSALF [31]), modified to contain a scFv (αMHC and αHEL, respectively [27]), upstream of the GP gene. Consequently, the resulting constructs (αMHC/VSV-G and αHEL/VSV-G) express the genes from the MLV LTR, with a MLV leader sequence (L mlv ) and 6 additional amino acids from the virus. (IV and V) Vectors for production of HIV-1 derived viral particles. A HIV-1-based lentiviral vector (V) (CD 416; pHRCMV) [29], into which EGFP gene had been inserted, together with a helper plasmid (CD 417; pCMV△R8.2) [29] expressing Gag, Pol and accessory HIV-1 proteins (IV), were used for production of HIV-1 particles. Expression vectors, physical maps and primer sequences are available upon request. b. VSV-G- and scFv/VSV-G pseudotyped HIV-1 particles, produced in 293T cells. 2 × 10 6 293T cells in 10 cm diameter tissue culture dishes were transiently transfected with 5 μg of an LTR-driven EGFP vector (pHRCMV-EGFP) and 4 μg of a helper plasmid (pCMV△R8.2) [29], using the calcium phosphate co-precipitation procedure [36]. 5 μg PM 730 or 30 μg scFv/VSV-G plasmid (PM 981 or PM 983) were also included. DNA precipitates were removed after 16 hours, and the viral supernatants were collected 24–48 hours later and pelleted by ultracentrifugation (BeckmanCoulter) at 25 kRPM, 4°C for 2 hours and resuspended in 1% of the original volume. Cell lysates and 2 μl concentrated scFv/VSV-G virus or 10 μl of non-concentrated VSV-G virus were separated on a 12 % SDS polyacrylamide gel and transferred onto <t>Protran</t> nitrocellulose membranes (Schleicher and Schuell). HA-tagged VSV-G and scFv/VSV-G were detected using a rat anti-HA antibody (Sigma), followed by horse radish peroxidase (HPO) conjugated anti-RatIgG (Dako). p24Gag was detected using SF2 rabbit monoclonal antibody (NIH AIDS Research and Reference Reagent Program) followed by anti-rabbit IgG/HPO (Santa Cruz), and was used as an internal reference to normalise for the virion protein quantities. The membranes were developed with Renaissance chemoluminescence kit (NEN Life Science Products), as recommended by the supplier.
    Nitrocellulose Membrane, supplied by Sartorius AG, used in various techniques. Bioz Stars score: 92/100, based on 280 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nitrocellulose membrane/product/Sartorius AG
    Average 92 stars, based on 280 article reviews
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    nitrocellulose membrane - by Bioz Stars, 2021-01
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    scFv-VSV-G expression plasmids and incorporation in HIV-1 derived particles. a) Expression plasmids: (I) VSV-G expression plasmid (PM 730). The 1.6 kB HindIII-BamHI VSV-G fragment (serotype Indiana) was transferred from pFB.VSVG (J.M. Heard, Paris, France) into pcDNA3 (InVitrogen) by PCR cloning according to standard procedures. A haemagglutinin (HA) sequence was added at the C terminus of VSV-G for immunodetection. (II and III) scFv/VSV-G expression plasmids. The chimaeric constructs were generated by PCR-based cloning. Mature VSV-G (from amino acid 17) was amplified from PM 730 and introduced into the PM 441 and PM 442 plasmids [23]. These constructs originate from an MLV-derived plasmid (FBMOSALF [31]), modified to contain a scFv (αMHC and αHEL, respectively [27]), upstream of the GP gene. Consequently, the resulting constructs (αMHC/VSV-G and αHEL/VSV-G) express the genes from the MLV LTR, with a MLV leader sequence (L mlv ) and 6 additional amino acids from the virus. (IV and V) Vectors for production of HIV-1 derived viral particles. A HIV-1-based lentiviral vector (V) (CD 416; pHRCMV) [29], into which EGFP gene had been inserted, together with a helper plasmid (CD 417; pCMV△R8.2) [29] expressing Gag, Pol and accessory HIV-1 proteins (IV), were used for production of HIV-1 particles. Expression vectors, physical maps and primer sequences are available upon request. b. VSV-G- and scFv/VSV-G pseudotyped HIV-1 particles, produced in 293T cells. 2 × 10 6 293T cells in 10 cm diameter tissue culture dishes were transiently transfected with 5 μg of an LTR-driven EGFP vector (pHRCMV-EGFP) and 4 μg of a helper plasmid (pCMV△R8.2) [29], using the calcium phosphate co-precipitation procedure [36]. 5 μg PM 730 or 30 μg scFv/VSV-G plasmid (PM 981 or PM 983) were also included. DNA precipitates were removed after 16 hours, and the viral supernatants were collected 24–48 hours later and pelleted by ultracentrifugation (BeckmanCoulter) at 25 kRPM, 4°C for 2 hours and resuspended in 1% of the original volume. Cell lysates and 2 μl concentrated scFv/VSV-G virus or 10 μl of non-concentrated VSV-G virus were separated on a 12 % SDS polyacrylamide gel and transferred onto <t>Protran</t> nitrocellulose membranes (Schleicher and Schuell). HA-tagged VSV-G and scFv/VSV-G were detected using a rat anti-HA antibody (Sigma), followed by horse radish peroxidase (HPO) conjugated anti-RatIgG (Dako). p24Gag was detected using SF2 rabbit monoclonal antibody (NIH AIDS Research and Reference Reagent Program) followed by anti-rabbit IgG/HPO (Santa Cruz), and was used as an internal reference to normalise for the virion protein quantities. The membranes were developed with Renaissance chemoluminescence kit (NEN Life Science Products), as recommended by the supplier.
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    scFv-VSV-G expression plasmids and incorporation in HIV-1 derived particles. a) Expression plasmids: (I) VSV-G expression plasmid (PM 730). The 1.6 kB HindIII-BamHI VSV-G fragment (serotype Indiana) was transferred from pFB.VSVG (J.M. Heard, Paris, France) into pcDNA3 (InVitrogen) by PCR cloning according to standard procedures. A haemagglutinin (HA) sequence was added at the C terminus of VSV-G for immunodetection. (II and III) scFv/VSV-G expression plasmids. The chimaeric constructs were generated by PCR-based cloning. Mature VSV-G (from amino acid 17) was amplified from PM 730 and introduced into the PM 441 and PM 442 plasmids [23]. These constructs originate from an MLV-derived plasmid (FBMOSALF [31]), modified to contain a scFv (αMHC and αHEL, respectively [27]), upstream of the GP gene. Consequently, the resulting constructs (αMHC/VSV-G and αHEL/VSV-G) express the genes from the MLV LTR, with a MLV leader sequence (L mlv ) and 6 additional amino acids from the virus. (IV and V) Vectors for production of HIV-1 derived viral particles. A HIV-1-based lentiviral vector (V) (CD 416; pHRCMV) [29], into which EGFP gene had been inserted, together with a helper plasmid (CD 417; pCMV△R8.2) [29] expressing Gag, Pol and accessory HIV-1 proteins (IV), were used for production of HIV-1 particles. Expression vectors, physical maps and primer sequences are available upon request. b. VSV-G- and scFv/VSV-G pseudotyped HIV-1 particles, produced in 293T cells. 2 × 10 6 293T cells in 10 cm diameter tissue culture dishes were transiently transfected with 5 μg of an LTR-driven EGFP vector (pHRCMV-EGFP) and 4 μg of a helper plasmid (pCMV△R8.2) [29], using the calcium phosphate co-precipitation procedure [36]. 5 μg PM 730 or 30 μg scFv/VSV-G plasmid (PM 981 or PM 983) were also included. DNA precipitates were removed after 16 hours, and the viral supernatants were collected 24–48 hours later and pelleted by ultracentrifugation (BeckmanCoulter) at 25 kRPM, 4°C for 2 hours and resuspended in 1% of the original volume. Cell lysates and 2 μl concentrated scFv/VSV-G virus or 10 μl of non-concentrated VSV-G virus were separated on a 12 % SDS polyacrylamide gel and transferred onto Protran nitrocellulose membranes (Schleicher and Schuell). HA-tagged VSV-G and scFv/VSV-G were detected using a rat anti-HA antibody (Sigma), followed by horse radish peroxidase (HPO) conjugated anti-RatIgG (Dako). p24Gag was detected using SF2 rabbit monoclonal antibody (NIH AIDS Research and Reference Reagent Program) followed by anti-rabbit IgG/HPO (Santa Cruz), and was used as an internal reference to normalise for the virion protein quantities. The membranes were developed with Renaissance chemoluminescence kit (NEN Life Science Products), as recommended by the supplier.

    Journal: Virology Journal

    Article Title: The effects of N-terminal insertion into VSV-G of an scFv peptide

    doi: 10.1186/1743-422X-3-69

    Figure Lengend Snippet: scFv-VSV-G expression plasmids and incorporation in HIV-1 derived particles. a) Expression plasmids: (I) VSV-G expression plasmid (PM 730). The 1.6 kB HindIII-BamHI VSV-G fragment (serotype Indiana) was transferred from pFB.VSVG (J.M. Heard, Paris, France) into pcDNA3 (InVitrogen) by PCR cloning according to standard procedures. A haemagglutinin (HA) sequence was added at the C terminus of VSV-G for immunodetection. (II and III) scFv/VSV-G expression plasmids. The chimaeric constructs were generated by PCR-based cloning. Mature VSV-G (from amino acid 17) was amplified from PM 730 and introduced into the PM 441 and PM 442 plasmids [23]. These constructs originate from an MLV-derived plasmid (FBMOSALF [31]), modified to contain a scFv (αMHC and αHEL, respectively [27]), upstream of the GP gene. Consequently, the resulting constructs (αMHC/VSV-G and αHEL/VSV-G) express the genes from the MLV LTR, with a MLV leader sequence (L mlv ) and 6 additional amino acids from the virus. (IV and V) Vectors for production of HIV-1 derived viral particles. A HIV-1-based lentiviral vector (V) (CD 416; pHRCMV) [29], into which EGFP gene had been inserted, together with a helper plasmid (CD 417; pCMV△R8.2) [29] expressing Gag, Pol and accessory HIV-1 proteins (IV), were used for production of HIV-1 particles. Expression vectors, physical maps and primer sequences are available upon request. b. VSV-G- and scFv/VSV-G pseudotyped HIV-1 particles, produced in 293T cells. 2 × 10 6 293T cells in 10 cm diameter tissue culture dishes were transiently transfected with 5 μg of an LTR-driven EGFP vector (pHRCMV-EGFP) and 4 μg of a helper plasmid (pCMV△R8.2) [29], using the calcium phosphate co-precipitation procedure [36]. 5 μg PM 730 or 30 μg scFv/VSV-G plasmid (PM 981 or PM 983) were also included. DNA precipitates were removed after 16 hours, and the viral supernatants were collected 24–48 hours later and pelleted by ultracentrifugation (BeckmanCoulter) at 25 kRPM, 4°C for 2 hours and resuspended in 1% of the original volume. Cell lysates and 2 μl concentrated scFv/VSV-G virus or 10 μl of non-concentrated VSV-G virus were separated on a 12 % SDS polyacrylamide gel and transferred onto Protran nitrocellulose membranes (Schleicher and Schuell). HA-tagged VSV-G and scFv/VSV-G were detected using a rat anti-HA antibody (Sigma), followed by horse radish peroxidase (HPO) conjugated anti-RatIgG (Dako). p24Gag was detected using SF2 rabbit monoclonal antibody (NIH AIDS Research and Reference Reagent Program) followed by anti-rabbit IgG/HPO (Santa Cruz), and was used as an internal reference to normalise for the virion protein quantities. The membranes were developed with Renaissance chemoluminescence kit (NEN Life Science Products), as recommended by the supplier.

    Article Snippet: The samples were fractionated through SDS polyacrylamide (10%) gels (SDS-PAGE) and transferred to Protran nitrocellulose membranes (Schleicher and Schuell).

    Techniques: Expressing, Derivative Assay, Plasmid Preparation, Polymerase Chain Reaction, Clone Assay, Sequencing, Immunodetection, Construct, Generated, Amplification, Modification, Produced, Transfection