Binding Assay:Article Title: Generation of Conformation‐Specific Monoclonal Antibodies for Integral Membrane Proteins
Article Snippet: .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination. .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination.
Selection:Article Title: Generation of Conformation‐Specific Monoclonal Antibodies for Integral Membrane Proteins
Article Snippet: .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination. .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination.
Inverted Microscopy:Article Title: Generation of Conformation‐Specific Monoclonal Antibodies for Integral Membrane Proteins
Article Snippet: .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination. .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination.
Chromatography:Article Title: Generation of Conformation‐Specific Monoclonal Antibodies for Integral Membrane Proteins
Article Snippet: .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination. .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination.
Cloning:Article Title: Generation of Conformation‐Specific Monoclonal Antibodies for Integral Membrane Proteins
Article Snippet: .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination. .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination.
Transformation Assay:Article Title: Generation of Conformation‐Specific Monoclonal Antibodies for Integral Membrane Proteins
Article Snippet: .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination. .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination.
Polymerase Chain Reaction:Article Title: Generation of Conformation‐Specific Monoclonal Antibodies for Integral Membrane Proteins
Article Snippet: .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination. .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination.
Transfection:Article Title: Generation of Conformation‐Specific Monoclonal Antibodies for Integral Membrane Proteins
Article Snippet: .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination. .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination.
Virus:Article Title: Generation of Conformation‐Specific Monoclonal Antibodies for Integral Membrane Proteins
Article Snippet: .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination. .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination.
Amplification:Article Title: Generation of Conformation‐Specific Monoclonal Antibodies for Integral Membrane Proteins
Article Snippet: .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination. .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination.
Endpoint Dilution Assay:Article Title: Generation of Conformation‐Specific Monoclonal Antibodies for Integral Membrane Proteins
Article Snippet: .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination. .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination.
Filtration:Article Title: Generation of Conformation‐Specific Monoclonal Antibodies for Integral Membrane Proteins
Article Snippet: .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination. .. Hybridoma cells (see Basic Protocol ) Hybridoma‐SFM (Thermo Fisher Scientific, cat. no. 12045076), 37°C Hybridoma growth medium (see recipe), 37°C Trypan blue (Corning/Cellgro, cat. no. 25‐900‐CL) Protein A/G agarose (Pierce, cat. no, 20422) Protein A IgG binding buffer (Pierce, cat. no. 21007) Protein A IgG elution buffer (Pierce, cat. no. 21009) 1 M Tris buffer, pH 8.0 pFastBac Dual vector (Thermo Fisher Scientific, cat. no. 10712024) Escherichia coli DH10 Bac cells (Life Technologies, cat. no. 10361‐012) Bacmid selection plates (see recipe) SF9 cells (Thermo Fisher Scientific, cat. no. 12659017) SF900 III SFM (Thermo Fisher Scientific, cat. no. 1258027), 37°C Sf9 Easy Titer cell line (Kerafast, cat. no. ENH022‐FP) TBS (see recipe) supplemented with 5 mM desthiobiotin (IBA Lifesciences, cat. no. 2‐1000‐002) Liquid nitrogen Sorvall Legend XFR (Thermo Fisher Scientific, cat. no. 75004539) CELLine bioreactor flask (Corning, cat. no. 353137) Hemacytometer (Hausser Scientific, cat. no. 1492) Inverted microscope (Nikon Eclipse TS100) Econo‐Pac chromatography columns (Bio‐Rad, cat. no. 7321010) 50‐ml conical tubes Flat‐bottom Erlenmeyer flasks (Greiner Bio‐One, cat. no. 679505) 27°C incubator shaker (Innova 44R; Eppendorf New Brunswick Scientific, cat. no. M1282‐0004) Strep‐Tactin 4Flow high‐capacity resin (IBA Lifesciences, cat. no. 2‐1250‐010) equilibrated with TBS (see recipe) 30‐kDa‐cutoff filters (Amicon Ultra Centrifugal Filter, 30 kDa MWCO, Millipore Sigma, cat. no. UFC9030) Superdex 200 Increase 10/300 GL column (Cytiva, cat. no. 28990944) equilibrated with TBS (see recipe) Additional reagents and equipment for cell freezing (see Basic Protocol , step 79); cloning, transformation, and selection; PCR; Sf9 transfection and virus amplification (Goehring et al., ); endpoint dilution assay (Goehring et al., ); and supernatant collection and tangential flow filtration (see Support Protocol ) NOTE : Experiments involving PCR require extremely careful technique to prevent contamination.
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