Structured Review

Roche phenylmethylsulfonyl fluoride pmsf
Phenylmethylsulfonyl Fluoride Pmsf, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/phenylmethylsulfonyl fluoride pmsf/product/Roche
Average 94 stars, based on 190 article reviews
Price from $9.99 to $1999.99
phenylmethylsulfonyl fluoride pmsf - by Bioz Stars, 2020-05
94/100 stars

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Centrifugation:

Article Title: Steroidogenic Factor 1 (NR5A1) Maintains Centrosome Homeostasis in Steroidogenic Cells by Restricting Centrosomal DNA-Dependent Protein Kinase Activation
Article Snippet: .. Sucrose fraction 5 was agitated in 20 ml of extraction buffer containing 20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), 2 mM phenylmethylsulfonyl fluoride (PMSF), and the protease and phosphatase inhibitor cocktail (Roche, Mannheim, Germany) on ice for 30 min, followed by centrifugation at 15,000 rpm at 4°C for 10 min. .. The supernatant was concentrated on an Amicon filter (10-kDa cutoff, 5% bovine serum albumin [BSA] preblocked; Millipore, MA) by centrifugation at 4,000 × g at 4°C for 1 h. The aggregate in the protein concentrate was removed by centrifugation at 15,000 rpm for 30 min, and the supernatant was used as centrosome extracts.

Article Title: Ubiquitin-Independent Degradation of Antiapoptotic MCL-1 ▿
Article Snippet: .. For immunoblot analysis, cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing 1 mM NaF, 100 μM phenylmethylsulfonyl fluoride (PMSF), and 200 μM Na3 VO4 supplemented with complete EDTA-free protease inhibitors (Roche) on ice for 30 min. Lysates were cleared by centrifugation, and protein concentrations were determined by bicinchoninic acid (BCA) protein assay. .. For coimmunoprecipitation studies, wild-type MEFs, Mcl-1 -deleted MEFs alone, or MEFs stably expressing tagless MCL-1 or MCL-1KR were lysed in flag lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100, 1 mM EDTA) containing protease inhibitors on ice for 30 min.

Radio Immunoprecipitation:

Article Title: Ubiquitin-Independent Degradation of Antiapoptotic MCL-1 ▿
Article Snippet: .. For immunoblot analysis, cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing 1 mM NaF, 100 μM phenylmethylsulfonyl fluoride (PMSF), and 200 μM Na3 VO4 supplemented with complete EDTA-free protease inhibitors (Roche) on ice for 30 min. Lysates were cleared by centrifugation, and protein concentrations were determined by bicinchoninic acid (BCA) protein assay. .. For coimmunoprecipitation studies, wild-type MEFs, Mcl-1 -deleted MEFs alone, or MEFs stably expressing tagless MCL-1 or MCL-1KR were lysed in flag lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100, 1 mM EDTA) containing protease inhibitors on ice for 30 min.

Article Title: Histone Demethylase JMJD2A Regulates Kaposi's Sarcoma-Associated Herpesvirus Replication and Is Targeted by a Viral Transcriptional Factor ▿
Article Snippet: .. Transfected 293T cells were collected in modified radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 6.7], 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1× protease inhibitor cocktail (Roche). .. Total cell lysates (TCLs) were incubated with either mouse nonimmune serum IgG, rabbit nonimmune serum IgG, anti-Flag M2 agarose (Sigma), or anti-JMJD2A antibody overnight at 4°C.

Protease Inhibitor:

Article Title: Hemoglobin Subunit Beta Interacts with the Capsid Protein and Antagonizes the Growth of Classical Swine Fever Virus
Article Snippet: .. The transfected cells were harvested at 48 hpt, washed three times with cold PBS (pH 7.4), and lysed with NP-40 buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 0.5% NP-40, 0.5 mM EDTA) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mg/ml protease inhibitor cocktail (Roche) at 4°C for 1 h. Clarified extracts were precleared with protein A/G beads (SC-2003; Santa Cruz) and then incubated with protein A/G beads plus anti-Flag monoclonal antibody (MAb) F3040 (Sigma) for 4 h. The beads were then washed with NP-40 buffer and boiled in sample buffer, and the proteins were subjected to SDS-PAGE, followed by immunoblotting analysis with anti-Flag (F7425; Sigma) and anti-Myc polyclonal antibodies (pAb) (E022050-1; Earthox LLC). .. Following the indicated treatment, total RNA was extracted from CSFV-infected cells with TRIzol (15596026; Invitrogen) and treated with DNase I to remove potential genomic DNA contamination.

Article Title: Spn1 Regulates the GNBP3-Dependent Toll Signaling Pathway in Drosophila melanogaster ▿
Article Snippet: .. Bacteria were sonicated in 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 0.5 M EDTA buffer containing 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and complete inhibitors (protease inhibitor cocktail tablets; Roche); samples were centrifuged at 16,400 rpm for 30 min and loaded onto a chitin column. .. Protein was concentrated with a 30,000-nominal-molecular-weight-limit (NMWL) membrane Amicon centrifugal filter (Millipore) in 20 mM Tris-HCl, pH 7.5, buffer, using an ÄKTA-FLPC fast protein liquid chromatography system.

Article Title: In Vivo Imaging of Diacylglycerol at the Cytoplasmic Leaflet of Plant Membranes
Article Snippet: .. Roots of 11-day old seedlings were ground in liquid nitrogen and proteins were extracted using 2 ✕ (w/v) protein extraction buffer [0.5% (v/v) NP-40, 75 mM NaCl, 100 mM Tris–HCl pH 8.0, 1% (w/v) polyvinylpolypyrrolidone (PVPP), 100 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM EDTA (pH 8.0) and complete, EDTA-free Protease Inhibitor Cocktail (Roche)]. ..

Article Title: Histone Demethylase JMJD2A Regulates Kaposi's Sarcoma-Associated Herpesvirus Replication and Is Targeted by a Viral Transcriptional Factor ▿
Article Snippet: .. Transfected 293T cells were collected in modified radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 6.7], 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1× protease inhibitor cocktail (Roche). .. Total cell lysates (TCLs) were incubated with either mouse nonimmune serum IgG, rabbit nonimmune serum IgG, anti-Flag M2 agarose (Sigma), or anti-JMJD2A antibody overnight at 4°C.

Article Title: N-terminus of the rat adenine glycosylase MYH affects excision rates and processing of MYH-generated abasic sites
Article Snippet: .. Briefly, cell pellets were suspended in cold hypotonic buffer (10 mM HEPES, pH 7.6, 10 mM KCl, 0.1 mM EDTA, 0.1 mM ethyleneglycol-bis(aminoethylether)-tetraacetic acid (EGTA), 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 μg/ml Pepstatin supplemented with the Complete Protease Inhibitor Cocktail Tablet (Roche Diagnostics) and incubated on ice for 30 min with occasional mixing. ..

Article Title: Cytosolic Phospholipase A2 Gamma Is Involved in Hepatitis C Virus Replication and Assembly
Article Snippet: .. Cells were lysed in immunoprecipitation (IP) buffer containing 50 mM Tris, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 150 mM NaCl, 100 μM phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail (Complete Mini; Roche) for 30 min. ..

Transfection:

Article Title: Hemoglobin Subunit Beta Interacts with the Capsid Protein and Antagonizes the Growth of Classical Swine Fever Virus
Article Snippet: .. The transfected cells were harvested at 48 hpt, washed three times with cold PBS (pH 7.4), and lysed with NP-40 buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 0.5% NP-40, 0.5 mM EDTA) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mg/ml protease inhibitor cocktail (Roche) at 4°C for 1 h. Clarified extracts were precleared with protein A/G beads (SC-2003; Santa Cruz) and then incubated with protein A/G beads plus anti-Flag monoclonal antibody (MAb) F3040 (Sigma) for 4 h. The beads were then washed with NP-40 buffer and boiled in sample buffer, and the proteins were subjected to SDS-PAGE, followed by immunoblotting analysis with anti-Flag (F7425; Sigma) and anti-Myc polyclonal antibodies (pAb) (E022050-1; Earthox LLC). .. Following the indicated treatment, total RNA was extracted from CSFV-infected cells with TRIzol (15596026; Invitrogen) and treated with DNase I to remove potential genomic DNA contamination.

Article Title: Histone Demethylase JMJD2A Regulates Kaposi's Sarcoma-Associated Herpesvirus Replication and Is Targeted by a Viral Transcriptional Factor ▿
Article Snippet: .. Transfected 293T cells were collected in modified radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 6.7], 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1× protease inhibitor cocktail (Roche). .. Total cell lysates (TCLs) were incubated with either mouse nonimmune serum IgG, rabbit nonimmune serum IgG, anti-Flag M2 agarose (Sigma), or anti-JMJD2A antibody overnight at 4°C.

Incubation:

Article Title: Hemoglobin Subunit Beta Interacts with the Capsid Protein and Antagonizes the Growth of Classical Swine Fever Virus
Article Snippet: .. The transfected cells were harvested at 48 hpt, washed three times with cold PBS (pH 7.4), and lysed with NP-40 buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 0.5% NP-40, 0.5 mM EDTA) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mg/ml protease inhibitor cocktail (Roche) at 4°C for 1 h. Clarified extracts were precleared with protein A/G beads (SC-2003; Santa Cruz) and then incubated with protein A/G beads plus anti-Flag monoclonal antibody (MAb) F3040 (Sigma) for 4 h. The beads were then washed with NP-40 buffer and boiled in sample buffer, and the proteins were subjected to SDS-PAGE, followed by immunoblotting analysis with anti-Flag (F7425; Sigma) and anti-Myc polyclonal antibodies (pAb) (E022050-1; Earthox LLC). .. Following the indicated treatment, total RNA was extracted from CSFV-infected cells with TRIzol (15596026; Invitrogen) and treated with DNase I to remove potential genomic DNA contamination.

Article Title: N-terminus of the rat adenine glycosylase MYH affects excision rates and processing of MYH-generated abasic sites
Article Snippet: .. Briefly, cell pellets were suspended in cold hypotonic buffer (10 mM HEPES, pH 7.6, 10 mM KCl, 0.1 mM EDTA, 0.1 mM ethyleneglycol-bis(aminoethylether)-tetraacetic acid (EGTA), 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 μg/ml Pepstatin supplemented with the Complete Protease Inhibitor Cocktail Tablet (Roche Diagnostics) and incubated on ice for 30 min with occasional mixing. ..

Immunoprecipitation:

Article Title: Cytosolic Phospholipase A2 Gamma Is Involved in Hepatitis C Virus Replication and Assembly
Article Snippet: .. Cells were lysed in immunoprecipitation (IP) buffer containing 50 mM Tris, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 150 mM NaCl, 100 μM phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail (Complete Mini; Roche) for 30 min. ..

Protein Extraction:

Article Title: In Vivo Imaging of Diacylglycerol at the Cytoplasmic Leaflet of Plant Membranes
Article Snippet: .. Roots of 11-day old seedlings were ground in liquid nitrogen and proteins were extracted using 2 ✕ (w/v) protein extraction buffer [0.5% (v/v) NP-40, 75 mM NaCl, 100 mM Tris–HCl pH 8.0, 1% (w/v) polyvinylpolypyrrolidone (PVPP), 100 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM EDTA (pH 8.0) and complete, EDTA-free Protease Inhibitor Cocktail (Roche)]. ..

BIA-KA:

Article Title: Ubiquitin-Independent Degradation of Antiapoptotic MCL-1 ▿
Article Snippet: .. For immunoblot analysis, cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing 1 mM NaF, 100 μM phenylmethylsulfonyl fluoride (PMSF), and 200 μM Na3 VO4 supplemented with complete EDTA-free protease inhibitors (Roche) on ice for 30 min. Lysates were cleared by centrifugation, and protein concentrations were determined by bicinchoninic acid (BCA) protein assay. .. For coimmunoprecipitation studies, wild-type MEFs, Mcl-1 -deleted MEFs alone, or MEFs stably expressing tagless MCL-1 or MCL-1KR were lysed in flag lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100, 1 mM EDTA) containing protease inhibitors on ice for 30 min.

Modification:

Article Title: Histone Demethylase JMJD2A Regulates Kaposi's Sarcoma-Associated Herpesvirus Replication and Is Targeted by a Viral Transcriptional Factor ▿
Article Snippet: .. Transfected 293T cells were collected in modified radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl [pH 6.7], 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1× protease inhibitor cocktail (Roche). .. Total cell lysates (TCLs) were incubated with either mouse nonimmune serum IgG, rabbit nonimmune serum IgG, anti-Flag M2 agarose (Sigma), or anti-JMJD2A antibody overnight at 4°C.

Sonication:

Article Title: Spn1 Regulates the GNBP3-Dependent Toll Signaling Pathway in Drosophila melanogaster ▿
Article Snippet: .. Bacteria were sonicated in 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 0.5 M EDTA buffer containing 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and complete inhibitors (protease inhibitor cocktail tablets; Roche); samples were centrifuged at 16,400 rpm for 30 min and loaded onto a chitin column. .. Protein was concentrated with a 30,000-nominal-molecular-weight-limit (NMWL) membrane Amicon centrifugal filter (Millipore) in 20 mM Tris-HCl, pH 7.5, buffer, using an ÄKTA-FLPC fast protein liquid chromatography system.

SDS Page:

Article Title: Hemoglobin Subunit Beta Interacts with the Capsid Protein and Antagonizes the Growth of Classical Swine Fever Virus
Article Snippet: .. The transfected cells were harvested at 48 hpt, washed three times with cold PBS (pH 7.4), and lysed with NP-40 buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 0.5% NP-40, 0.5 mM EDTA) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mg/ml protease inhibitor cocktail (Roche) at 4°C for 1 h. Clarified extracts were precleared with protein A/G beads (SC-2003; Santa Cruz) and then incubated with protein A/G beads plus anti-Flag monoclonal antibody (MAb) F3040 (Sigma) for 4 h. The beads were then washed with NP-40 buffer and boiled in sample buffer, and the proteins were subjected to SDS-PAGE, followed by immunoblotting analysis with anti-Flag (F7425; Sigma) and anti-Myc polyclonal antibodies (pAb) (E022050-1; Earthox LLC). .. Following the indicated treatment, total RNA was extracted from CSFV-infected cells with TRIzol (15596026; Invitrogen) and treated with DNase I to remove potential genomic DNA contamination.

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    Roche pmsf treated roche dnasegii
    Proteases impair CD4 T cell responses to alum plus OVA-NP. ( A and B ) <t>Roche-DNaseGII</t> was treated with <t>PMSF</t> and assessed for (A) protease activity at indicated concentrations using a Pierce protease assay kit and (B) DNase enzymatic activity as described
    Pmsf Treated Roche Dnasegii, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pmsf treated roche dnasegii/product/Roche
    Average 90 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    pmsf treated roche dnasegii - by Bioz Stars, 2020-05
    90/100 stars
      Buy from Supplier

    88
    Roche gfp trap lysis buffer
    Immunoblot for <t>GFP,</t> actin, and PP1 of GFP-Trap pull-downs and 2% of input. HEK293T cells were transiently transfected with plasmids encoding the indicated constructs. After 36 hr, cells were lysed in GFP-Trap lysis buffer (150 mM <t>NaCl,</t> 10 mM Tris/Cl pH 7.5, 0.5 mM EDTA, 1 mM PMSF, and Protease Inhibitor Cocktail [Roche]) and post-nuclear supernatants were incubated with GFP-Trap beads at 4°C for 2 hr then washed four times in the same buffer. Next, samples were washed thrice in GFP-Trap lysis buffer supplemented with additional NaCl as indicated. DOI: http://dx.doi.org/10.7554/eLife.04872.008
    Gfp Trap Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gfp trap lysis buffer/product/Roche
    Average 88 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    gfp trap lysis buffer - by Bioz Stars, 2020-05
    88/100 stars
      Buy from Supplier

    Image Search Results


    Proteases impair CD4 T cell responses to alum plus OVA-NP. ( A and B ) Roche-DNaseGII was treated with PMSF and assessed for (A) protease activity at indicated concentrations using a Pierce protease assay kit and (B) DNase enzymatic activity as described

    Journal: The Journal of Immunology Author Choice

    Article Title: Contamination of DNase Preparations Confounds Analysis of the Role of DNA in Alum-Adjuvanted Vaccines

    doi: 10.4049/jimmunol.1501565

    Figure Lengend Snippet: Proteases impair CD4 T cell responses to alum plus OVA-NP. ( A and B ) Roche-DNaseGII was treated with PMSF and assessed for (A) protease activity at indicated concentrations using a Pierce protease assay kit and (B) DNase enzymatic activity as described

    Article Snippet: This dampening effect was consistent between wtDNase, mutDNase, and Worth-DNase ( ) and is similar in magnitude to that seen with the PMSF-treated Roche-DNaseGII and, to a lesser extent, the Roche-rDNase ( , ).

    Techniques: Activity Assay, Protease Assay

    Immunoblot for GFP, actin, and PP1 of GFP-Trap pull-downs and 2% of input. HEK293T cells were transiently transfected with plasmids encoding the indicated constructs. After 36 hr, cells were lysed in GFP-Trap lysis buffer (150 mM NaCl, 10 mM Tris/Cl pH 7.5, 0.5 mM EDTA, 1 mM PMSF, and Protease Inhibitor Cocktail [Roche]) and post-nuclear supernatants were incubated with GFP-Trap beads at 4°C for 2 hr then washed four times in the same buffer. Next, samples were washed thrice in GFP-Trap lysis buffer supplemented with additional NaCl as indicated. DOI: http://dx.doi.org/10.7554/eLife.04872.008

    Journal: eLife

    Article Title: Actin dynamics tune the integrated stress response by regulating eukaryotic initiation factor 2α dephosphorylation

    doi: 10.7554/eLife.04872

    Figure Lengend Snippet: Immunoblot for GFP, actin, and PP1 of GFP-Trap pull-downs and 2% of input. HEK293T cells were transiently transfected with plasmids encoding the indicated constructs. After 36 hr, cells were lysed in GFP-Trap lysis buffer (150 mM NaCl, 10 mM Tris/Cl pH 7.5, 0.5 mM EDTA, 1 mM PMSF, and Protease Inhibitor Cocktail [Roche]) and post-nuclear supernatants were incubated with GFP-Trap beads at 4°C for 2 hr then washed four times in the same buffer. Next, samples were washed thrice in GFP-Trap lysis buffer supplemented with additional NaCl as indicated. DOI: http://dx.doi.org/10.7554/eLife.04872.008

    Article Snippet: Briefly, cells were lysed in GFP-Trap lysis buffer (150 mM NaCl, 10 mM Tris/Cl pH 7.5, 0.5 mM EDTA, 1 mM PMSF, and Protease Inhibitor Cocktail [Roche]) and post-nuclear supernatants were incubated with GFP-Trap beads at 4°C for 2 hr then washed four times in the same buffer.

    Techniques: Transfection, Construct, Lysis, Protease Inhibitor, Incubation

    Immunoblot for GFP, actin, and PP1 of GFP-Trap pull-downs and 2% of input. HEK293T cells were transiently transfected with plasmids encoding the indicated constructs. After 36 hr, cells were lysed in GFP-Trap lysis buffer (150 mM NaCl, 10 mM Tris/Cl pH 7.5, 0.5 mM EDTA, 1 mM PMSF, and Protease Inhibitor Cocktail [Roche]) and post-nuclear supernatants were incubated with GFP-Trap beads at 4°C for 2 hr then washed once in the same buffer. Next, samples were washed thrice with no detergent (GFP-Trap lysis buffer), triton buffer (150 mM NaCl, 10 nM HEPES pH 7.4, 0.5% vol/vol triton X-100), RIPA buffer (150 mM NaCl, 50 mM Tris HCl pH 7.4, 1% vol/vol NP40, 0.5% vol/vol sodium deoxycholate, 0.1% vol/vol SDS) or digitonin buffer (150 mM NaCl, 50 mM Tris HCl pH 7.4, 0.1% vol/vol digitonin). DOI: http://dx.doi.org/10.7554/eLife.04872.009

    Journal: eLife

    Article Title: Actin dynamics tune the integrated stress response by regulating eukaryotic initiation factor 2α dephosphorylation

    doi: 10.7554/eLife.04872

    Figure Lengend Snippet: Immunoblot for GFP, actin, and PP1 of GFP-Trap pull-downs and 2% of input. HEK293T cells were transiently transfected with plasmids encoding the indicated constructs. After 36 hr, cells were lysed in GFP-Trap lysis buffer (150 mM NaCl, 10 mM Tris/Cl pH 7.5, 0.5 mM EDTA, 1 mM PMSF, and Protease Inhibitor Cocktail [Roche]) and post-nuclear supernatants were incubated with GFP-Trap beads at 4°C for 2 hr then washed once in the same buffer. Next, samples were washed thrice with no detergent (GFP-Trap lysis buffer), triton buffer (150 mM NaCl, 10 nM HEPES pH 7.4, 0.5% vol/vol triton X-100), RIPA buffer (150 mM NaCl, 50 mM Tris HCl pH 7.4, 1% vol/vol NP40, 0.5% vol/vol sodium deoxycholate, 0.1% vol/vol SDS) or digitonin buffer (150 mM NaCl, 50 mM Tris HCl pH 7.4, 0.1% vol/vol digitonin). DOI: http://dx.doi.org/10.7554/eLife.04872.009

    Article Snippet: Briefly, cells were lysed in GFP-Trap lysis buffer (150 mM NaCl, 10 mM Tris/Cl pH 7.5, 0.5 mM EDTA, 1 mM PMSF, and Protease Inhibitor Cocktail [Roche]) and post-nuclear supernatants were incubated with GFP-Trap beads at 4°C for 2 hr then washed four times in the same buffer.

    Techniques: Transfection, Construct, Lysis, Protease Inhibitor, Incubation