complete protease inhibitor cocktail roche  (Roche)

 
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    Structured Review

    Roche complete protease inhibitor cocktail roche
    Complete Protease Inhibitor Cocktail Roche, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/complete protease inhibitor cocktail roche/product/Roche
    Average 93 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    complete protease inhibitor cocktail roche - by Bioz Stars, 2020-04
    93/100 stars

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    Transfection:

    Article Title: Mammalian RNA Decay Pathways Are Highly Specialized and Widely Linked to Translation.
    Article Snippet: .. KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-FLAG M2 Sigma Cat#F1804 Anti-FLAG M2 Dynabeads Sigma Cat#M8823 Dynabeads M280 streptavidin-coated beads Thermo Fisher Cat#11206D Dynabeads Protein G Thermo Fisher Cat# 10004D Streptavidin-HRP Sigma Cat#S2438 Rabbit anti-AVEN ProScience Cat#2417 Rabbit anti-MTR4 ThermoFisher Scientific Cat#PA557927 Rabbit anti-ATF4 D4B8 Cell Signaling Cat#mAb11815 Rabbit anti-Phospho-eIF2a Ser51 D9G8 Cell Signaling Cat#mAb3398 Rabbit anti-eIF4E Bethyl Laboratories Cat#A301-154A Rat anti-tubulin clone YL1/2 Abcam Cat#ab6160 Rat anti-HA Roche Cat#11867423001 Chemicals, Peptides, and Recombinant Proteins T4 DNA Ligase Sigma Cat#10716359001 DMEM GIBCO Cat#21969-035 Non-essential amino acids GIBCO Cat#11140035 100 mM Sodium pyruvate GIBCO Cat#11360070 200 mM L-glutamine GIBCO Cat#25030024 Fetal bovine serum GIBCO Cat#10270106 Beta-mercaptoethanol Sigma Cat#M-7522 Gelatin Sigma Cat#G-1890 Trypsin-EDTA GIBCO Cat#25300-054 Dulbecco’s PBS GIBCO Cat#14190 Trypsin (TPCK-treated) Sigma Cat#T8802 OptiMEM GIBCO Cat#31985070 Lipofectamine 3000 Transfection kit Invitrogen Cat#L3000015 cOmplete Protease Inhibitor Cocktail Roche Cat#11836145001 Proteinase K Roche Cat#3115879001 SuperScript III Life Technologies Cat#18080085 3xFLAG peptide Sigma Cat#F4799-25MG RNace-It Ribonuclease Cocktail Agilent Cat#400720 TSAP Thermosensitive Alkaline Phosphatase Promega Cat#M9910 RNasin Ribonuclease Inhibitor Promega Cat#N2115 Recombinant RNasin Ribonuclease Inhibitor Promega Cat#N2511 miR-cat 33 conversion oligo pack IDT N/A T4 RNA Ligase 1 (ssRNA Ligase) NEB Cat#M0204L T4 PNK, T4 polynucleotide kinase NEB Cat#M0201L Hybond-C Extra membrane GE Healthcare Cat#RPN303E Kodak BioMax MS autoradiography film Kodak Cat#8222648 MetaPhor agarose Lonza Cat#50180 NuPAGE 4–12% (wt/vol) polyacrylamide Bis-Tris gels Life Technologies Cat#NP0335 NuPAGE LDS sample buffer 4 3 Life Technologies Cat#NP0007 NuPAGE SDS-MOPS running buffer Life Technologies Cat#NP0001 (Continued on next page) e1 Molecular Cell 77, 1–15.e1–e13, March 19, 2020 .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER NuPage transfer buffer Life Technologies Cat#NP00061 MinElute Gel extraction kit QIAGEN Cat#28604 Proteinase K Roche Cat#03115836001 RNase H NEB Cat#M0297L TaKaRa long and accurate (LA) Taq Clontech Cat#RR002M g32P-ATP 0.5 mCi 18.5 MBq Spec act.

    Recombinant:

    Article Title: Mammalian RNA Decay Pathways Are Highly Specialized and Widely Linked to Translation.
    Article Snippet: .. KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-FLAG M2 Sigma Cat#F1804 Anti-FLAG M2 Dynabeads Sigma Cat#M8823 Dynabeads M280 streptavidin-coated beads Thermo Fisher Cat#11206D Dynabeads Protein G Thermo Fisher Cat# 10004D Streptavidin-HRP Sigma Cat#S2438 Rabbit anti-AVEN ProScience Cat#2417 Rabbit anti-MTR4 ThermoFisher Scientific Cat#PA557927 Rabbit anti-ATF4 D4B8 Cell Signaling Cat#mAb11815 Rabbit anti-Phospho-eIF2a Ser51 D9G8 Cell Signaling Cat#mAb3398 Rabbit anti-eIF4E Bethyl Laboratories Cat#A301-154A Rat anti-tubulin clone YL1/2 Abcam Cat#ab6160 Rat anti-HA Roche Cat#11867423001 Chemicals, Peptides, and Recombinant Proteins T4 DNA Ligase Sigma Cat#10716359001 DMEM GIBCO Cat#21969-035 Non-essential amino acids GIBCO Cat#11140035 100 mM Sodium pyruvate GIBCO Cat#11360070 200 mM L-glutamine GIBCO Cat#25030024 Fetal bovine serum GIBCO Cat#10270106 Beta-mercaptoethanol Sigma Cat#M-7522 Gelatin Sigma Cat#G-1890 Trypsin-EDTA GIBCO Cat#25300-054 Dulbecco’s PBS GIBCO Cat#14190 Trypsin (TPCK-treated) Sigma Cat#T8802 OptiMEM GIBCO Cat#31985070 Lipofectamine 3000 Transfection kit Invitrogen Cat#L3000015 cOmplete Protease Inhibitor Cocktail Roche Cat#11836145001 Proteinase K Roche Cat#3115879001 SuperScript III Life Technologies Cat#18080085 3xFLAG peptide Sigma Cat#F4799-25MG RNace-It Ribonuclease Cocktail Agilent Cat#400720 TSAP Thermosensitive Alkaline Phosphatase Promega Cat#M9910 RNasin Ribonuclease Inhibitor Promega Cat#N2115 Recombinant RNasin Ribonuclease Inhibitor Promega Cat#N2511 miR-cat 33 conversion oligo pack IDT N/A T4 RNA Ligase 1 (ssRNA Ligase) NEB Cat#M0204L T4 PNK, T4 polynucleotide kinase NEB Cat#M0201L Hybond-C Extra membrane GE Healthcare Cat#RPN303E Kodak BioMax MS autoradiography film Kodak Cat#8222648 MetaPhor agarose Lonza Cat#50180 NuPAGE 4–12% (wt/vol) polyacrylamide Bis-Tris gels Life Technologies Cat#NP0335 NuPAGE LDS sample buffer 4 3 Life Technologies Cat#NP0007 NuPAGE SDS-MOPS running buffer Life Technologies Cat#NP0001 (Continued on next page) e1 Molecular Cell 77, 1–15.e1–e13, March 19, 2020 .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER NuPage transfer buffer Life Technologies Cat#NP00061 MinElute Gel extraction kit QIAGEN Cat#28604 Proteinase K Roche Cat#03115836001 RNase H NEB Cat#M0297L TaKaRa long and accurate (LA) Taq Clontech Cat#RR002M g32P-ATP 0.5 mCi 18.5 MBq Spec act.

    Article Title: CenH3-Independent Kinetochore Assembly in Lepidoptera Requires CCAN, Including CENP-T.
    Article Snippet: .. Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. .. Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Mammalian RNA Decay Pathways Are Highly Specialized and Widely Linked to Translation.
    Article Snippet: .. KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-FLAG M2 Sigma Cat#F1804 Anti-FLAG M2 Dynabeads Sigma Cat#M8823 Dynabeads M280 streptavidin-coated beads Thermo Fisher Cat#11206D Dynabeads Protein G Thermo Fisher Cat# 10004D Streptavidin-HRP Sigma Cat#S2438 Rabbit anti-AVEN ProScience Cat#2417 Rabbit anti-MTR4 ThermoFisher Scientific Cat#PA557927 Rabbit anti-ATF4 D4B8 Cell Signaling Cat#mAb11815 Rabbit anti-Phospho-eIF2a Ser51 D9G8 Cell Signaling Cat#mAb3398 Rabbit anti-eIF4E Bethyl Laboratories Cat#A301-154A Rat anti-tubulin clone YL1/2 Abcam Cat#ab6160 Rat anti-HA Roche Cat#11867423001 Chemicals, Peptides, and Recombinant Proteins T4 DNA Ligase Sigma Cat#10716359001 DMEM GIBCO Cat#21969-035 Non-essential amino acids GIBCO Cat#11140035 100 mM Sodium pyruvate GIBCO Cat#11360070 200 mM L-glutamine GIBCO Cat#25030024 Fetal bovine serum GIBCO Cat#10270106 Beta-mercaptoethanol Sigma Cat#M-7522 Gelatin Sigma Cat#G-1890 Trypsin-EDTA GIBCO Cat#25300-054 Dulbecco’s PBS GIBCO Cat#14190 Trypsin (TPCK-treated) Sigma Cat#T8802 OptiMEM GIBCO Cat#31985070 Lipofectamine 3000 Transfection kit Invitrogen Cat#L3000015 cOmplete Protease Inhibitor Cocktail Roche Cat#11836145001 Proteinase K Roche Cat#3115879001 SuperScript III Life Technologies Cat#18080085 3xFLAG peptide Sigma Cat#F4799-25MG RNace-It Ribonuclease Cocktail Agilent Cat#400720 TSAP Thermosensitive Alkaline Phosphatase Promega Cat#M9910 RNasin Ribonuclease Inhibitor Promega Cat#N2115 Recombinant RNasin Ribonuclease Inhibitor Promega Cat#N2511 miR-cat 33 conversion oligo pack IDT N/A T4 RNA Ligase 1 (ssRNA Ligase) NEB Cat#M0204L T4 PNK, T4 polynucleotide kinase NEB Cat#M0201L Hybond-C Extra membrane GE Healthcare Cat#RPN303E Kodak BioMax MS autoradiography film Kodak Cat#8222648 MetaPhor agarose Lonza Cat#50180 NuPAGE 4–12% (wt/vol) polyacrylamide Bis-Tris gels Life Technologies Cat#NP0335 NuPAGE LDS sample buffer 4 3 Life Technologies Cat#NP0007 NuPAGE SDS-MOPS running buffer Life Technologies Cat#NP0001 (Continued on next page) e1 Molecular Cell 77, 1–15.e1–e13, March 19, 2020 .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER NuPage transfer buffer Life Technologies Cat#NP00061 MinElute Gel extraction kit QIAGEN Cat#28604 Proteinase K Roche Cat#03115836001 RNase H NEB Cat#M0297L TaKaRa long and accurate (LA) Taq Clontech Cat#RR002M g32P-ATP 0.5 mCi 18.5 MBq Spec act.

    Article Title: CenH3-Independent Kinetochore Assembly in Lepidoptera Requires CCAN, Including CENP-T.
    Article Snippet: .. Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. .. Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres.

    Protease Inhibitor:

    Article Title: Mammalian RNA Decay Pathways Are Highly Specialized and Widely Linked to Translation.
    Article Snippet: .. KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-FLAG M2 Sigma Cat#F1804 Anti-FLAG M2 Dynabeads Sigma Cat#M8823 Dynabeads M280 streptavidin-coated beads Thermo Fisher Cat#11206D Dynabeads Protein G Thermo Fisher Cat# 10004D Streptavidin-HRP Sigma Cat#S2438 Rabbit anti-AVEN ProScience Cat#2417 Rabbit anti-MTR4 ThermoFisher Scientific Cat#PA557927 Rabbit anti-ATF4 D4B8 Cell Signaling Cat#mAb11815 Rabbit anti-Phospho-eIF2a Ser51 D9G8 Cell Signaling Cat#mAb3398 Rabbit anti-eIF4E Bethyl Laboratories Cat#A301-154A Rat anti-tubulin clone YL1/2 Abcam Cat#ab6160 Rat anti-HA Roche Cat#11867423001 Chemicals, Peptides, and Recombinant Proteins T4 DNA Ligase Sigma Cat#10716359001 DMEM GIBCO Cat#21969-035 Non-essential amino acids GIBCO Cat#11140035 100 mM Sodium pyruvate GIBCO Cat#11360070 200 mM L-glutamine GIBCO Cat#25030024 Fetal bovine serum GIBCO Cat#10270106 Beta-mercaptoethanol Sigma Cat#M-7522 Gelatin Sigma Cat#G-1890 Trypsin-EDTA GIBCO Cat#25300-054 Dulbecco’s PBS GIBCO Cat#14190 Trypsin (TPCK-treated) Sigma Cat#T8802 OptiMEM GIBCO Cat#31985070 Lipofectamine 3000 Transfection kit Invitrogen Cat#L3000015 cOmplete Protease Inhibitor Cocktail Roche Cat#11836145001 Proteinase K Roche Cat#3115879001 SuperScript III Life Technologies Cat#18080085 3xFLAG peptide Sigma Cat#F4799-25MG RNace-It Ribonuclease Cocktail Agilent Cat#400720 TSAP Thermosensitive Alkaline Phosphatase Promega Cat#M9910 RNasin Ribonuclease Inhibitor Promega Cat#N2115 Recombinant RNasin Ribonuclease Inhibitor Promega Cat#N2511 miR-cat 33 conversion oligo pack IDT N/A T4 RNA Ligase 1 (ssRNA Ligase) NEB Cat#M0204L T4 PNK, T4 polynucleotide kinase NEB Cat#M0201L Hybond-C Extra membrane GE Healthcare Cat#RPN303E Kodak BioMax MS autoradiography film Kodak Cat#8222648 MetaPhor agarose Lonza Cat#50180 NuPAGE 4–12% (wt/vol) polyacrylamide Bis-Tris gels Life Technologies Cat#NP0335 NuPAGE LDS sample buffer 4 3 Life Technologies Cat#NP0007 NuPAGE SDS-MOPS running buffer Life Technologies Cat#NP0001 (Continued on next page) e1 Molecular Cell 77, 1–15.e1–e13, March 19, 2020 .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER NuPage transfer buffer Life Technologies Cat#NP00061 MinElute Gel extraction kit QIAGEN Cat#28604 Proteinase K Roche Cat#03115836001 RNase H NEB Cat#M0297L TaKaRa long and accurate (LA) Taq Clontech Cat#RR002M g32P-ATP 0.5 mCi 18.5 MBq Spec act.

    Article Title: Novel Potassium Channels in Kidney Mitochondria: The Hyperpolarization-Activated and Cyclic Nucleotide-Gated HCN Channels
    Article Snippet: .. Anti-HCN1, anti-HCN2, anti-HCN3 and anti-HCN4 were purchased from Alomone (Jerusalem, Israel). cOmplete™ Protease Inhibitor Cocktail Roche, Mexico. .. ZD7288 was purchased from Tocris (Minneapolis, MN, USA). pcDNA3-mHCN1, pcDNA3-mHCN2, pcDNA3-hHCN4 and pEGFP·C3 were provided by Luis Vaca. pcDNA3-mHCN3 was provided by Andreas Ludwig.

    Article Title: mRNA Decapping and 5′-3′ Decay Contribute to the Regulation of ABA Signaling in Arabidopsis thaliana
    Article Snippet: .. In-gel kinase assay Frozen seedlings were ground in liquid nitrogen with mortar and pestle and sonicated three times for 20 s in the extraction buffer (20 mM Tris, pH 7.5, 2 mM EDTA, 2 mM EGTA 50 mM β-glycerophosphate, 100 μM Na3 VO4 , 2 mM dithiothreitol [DTT], Complete protease inhibitor cocktail Roche) using approximately 0.5 mL of the extraction buffer for each 1 μg of plant material. .. After sonication, the extracts were centrifuged at 18,000 rcf for 30 min at 4°C, and the supernatants were used for further studies.

    Article Title: A single-dose plasmid-launched live-attenuated Zika vaccine induces protective immunity
    Article Snippet: .. Cells from the 6-well plates were washed once with PBS and lysed at 4 °C for 1 h in 200 μl RIPA lysis buffer (ThermoFisher Scientific) supplemented with 1× complete protease inhibitor cocktail (Roche). ..

    Article Title: The degradation of p53 and its major E3 ligase Mdm2 is differentially dependent on the proteasomal ubiquitin receptor S5a
    Article Snippet: .. Immunoprecipitation For proteasome immunoprecipitation, cells were washed twice in PBS and lysed in buffer 2: 20 mM Tris (pH 7.5), 0.1% NP-40, 5 mM MgCl2 , 10 mM NaF, 10 mM Na-pyrophosphate, 10 mM β-glycerophosphate, 20% glycerol, 2 mM ATP, 1 mM DTT, complete protease inhibitor cocktail Roche. ..

    Article Title: A single-dose plasmid-launched live-attenuated Zika vaccine induces protective immunity
    Article Snippet: .. 2.6 SDS-PAGE and western blot Cells from the 6-well plates were washed once with PBS and lysed at 4 °C for 1 h in 200 μl RIPA lysis buffer (ThermoFisher Scientific) supplemented with 1× complete protease inhibitor cocktail (Roche). ..

    Article Title: ADAP is an upstream regulator that precedes SLP-76 at sites of TCR engagement and stabilizes signaling microclusters
    Article Snippet: .. Stimulations were stopped at the indicated times by dilution into ice-cold PBS supplemented with 10†mM NaF and 1†mM Na3 VO4 , centrifuged, and lysed in ice cold buffer containing 20†mM Tris-HCl (pH 8.0), 150†mM NaCl, 1†mM EDTA, 1% Triton X-100, 10†mM NaF, 1†mM Na3 VO4 and Complete protease inhibitor cocktail (Roche). ..

    Article Title: CenH3-Independent Kinetochore Assembly in Lepidoptera Requires CCAN, Including CENP-T.
    Article Snippet: .. Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. .. Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres.

    Autoradiography:

    Article Title: Mammalian RNA Decay Pathways Are Highly Specialized and Widely Linked to Translation.
    Article Snippet: .. KEY RESOURCES TABLE REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse anti-FLAG M2 Sigma Cat#F1804 Anti-FLAG M2 Dynabeads Sigma Cat#M8823 Dynabeads M280 streptavidin-coated beads Thermo Fisher Cat#11206D Dynabeads Protein G Thermo Fisher Cat# 10004D Streptavidin-HRP Sigma Cat#S2438 Rabbit anti-AVEN ProScience Cat#2417 Rabbit anti-MTR4 ThermoFisher Scientific Cat#PA557927 Rabbit anti-ATF4 D4B8 Cell Signaling Cat#mAb11815 Rabbit anti-Phospho-eIF2a Ser51 D9G8 Cell Signaling Cat#mAb3398 Rabbit anti-eIF4E Bethyl Laboratories Cat#A301-154A Rat anti-tubulin clone YL1/2 Abcam Cat#ab6160 Rat anti-HA Roche Cat#11867423001 Chemicals, Peptides, and Recombinant Proteins T4 DNA Ligase Sigma Cat#10716359001 DMEM GIBCO Cat#21969-035 Non-essential amino acids GIBCO Cat#11140035 100 mM Sodium pyruvate GIBCO Cat#11360070 200 mM L-glutamine GIBCO Cat#25030024 Fetal bovine serum GIBCO Cat#10270106 Beta-mercaptoethanol Sigma Cat#M-7522 Gelatin Sigma Cat#G-1890 Trypsin-EDTA GIBCO Cat#25300-054 Dulbecco’s PBS GIBCO Cat#14190 Trypsin (TPCK-treated) Sigma Cat#T8802 OptiMEM GIBCO Cat#31985070 Lipofectamine 3000 Transfection kit Invitrogen Cat#L3000015 cOmplete Protease Inhibitor Cocktail Roche Cat#11836145001 Proteinase K Roche Cat#3115879001 SuperScript III Life Technologies Cat#18080085 3xFLAG peptide Sigma Cat#F4799-25MG RNace-It Ribonuclease Cocktail Agilent Cat#400720 TSAP Thermosensitive Alkaline Phosphatase Promega Cat#M9910 RNasin Ribonuclease Inhibitor Promega Cat#N2115 Recombinant RNasin Ribonuclease Inhibitor Promega Cat#N2511 miR-cat 33 conversion oligo pack IDT N/A T4 RNA Ligase 1 (ssRNA Ligase) NEB Cat#M0204L T4 PNK, T4 polynucleotide kinase NEB Cat#M0201L Hybond-C Extra membrane GE Healthcare Cat#RPN303E Kodak BioMax MS autoradiography film Kodak Cat#8222648 MetaPhor agarose Lonza Cat#50180 NuPAGE 4–12% (wt/vol) polyacrylamide Bis-Tris gels Life Technologies Cat#NP0335 NuPAGE LDS sample buffer 4 3 Life Technologies Cat#NP0007 NuPAGE SDS-MOPS running buffer Life Technologies Cat#NP0001 (Continued on next page) e1 Molecular Cell 77, 1–15.e1–e13, March 19, 2020 .. Continued REAGENT or RESOURCE SOURCE IDENTIFIER NuPage transfer buffer Life Technologies Cat#NP00061 MinElute Gel extraction kit QIAGEN Cat#28604 Proteinase K Roche Cat#03115836001 RNase H NEB Cat#M0297L TaKaRa long and accurate (LA) Taq Clontech Cat#RR002M g32P-ATP 0.5 mCi 18.5 MBq Spec act.

    Blocking Assay:

    Article Title: CenH3-Independent Kinetochore Assembly in Lepidoptera Requires CCAN, Including CENP-T.
    Article Snippet: .. Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. .. Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres.

    Immunoprecipitation:

    Article Title: The degradation of p53 and its major E3 ligase Mdm2 is differentially dependent on the proteasomal ubiquitin receptor S5a
    Article Snippet: .. Immunoprecipitation For proteasome immunoprecipitation, cells were washed twice in PBS and lysed in buffer 2: 20 mM Tris (pH 7.5), 0.1% NP-40, 5 mM MgCl2 , 10 mM NaF, 10 mM Na-pyrophosphate, 10 mM β-glycerophosphate, 20% glycerol, 2 mM ATP, 1 mM DTT, complete protease inhibitor cocktail Roche. ..

    Incubation:

    Article Title: The degradation of p53 and its major E3 ligase Mdm2 is differentially dependent on the proteasomal ubiquitin receptor S5a
    Article Snippet: Immunoprecipitation For proteasome immunoprecipitation, cells were washed twice in PBS and lysed in buffer 2: 20 mM Tris (pH 7.5), 0.1% NP-40, 5 mM MgCl2 , 10 mM NaF, 10 mM Na-pyrophosphate, 10 mM β-glycerophosphate, 20% glycerol, 2 mM ATP, 1 mM DTT, complete protease inhibitor cocktail Roche. .. The pooled supernatants from one six-well plate (total volume 350 μl) were then incubated for 1 h at 4 °C with agarose beads covalently coupled to control immunoglobulin G, anti- PSMA2 antibody (MCP21, PW8335, Enzo Life Sciences) or anti-HA antibody (26181, Thermo Fisher Scientific).

    Article Title: ADAP is an upstream regulator that precedes SLP-76 at sites of TCR engagement and stabilizes signaling microclusters
    Article Snippet: Stimulations were stopped at the indicated times by dilution into ice-cold PBS supplemented with 10†mM NaF and 1†mM Na3 VO4 , centrifuged, and lysed in ice cold buffer containing 20†mM Tris-HCl (pH 8.0), 150†mM NaCl, 1†mM EDTA, 1% Triton X-100, 10†mM NaF, 1†mM Na3 VO4 and Complete protease inhibitor cocktail (Roche). .. For immunoprecipitations, cleared lysates were incubated with the indicated antibodies pre-bound to protein G or protein A beads (Thermo Fisher) for 1–2†h at room temperature.

    Activity Assay:

    Article Title: mRNA Decapping and 5′-3′ Decay Contribute to the Regulation of ABA Signaling in Arabidopsis thaliana
    Article Snippet: In-gel kinase assay Frozen seedlings were ground in liquid nitrogen with mortar and pestle and sonicated three times for 20 s in the extraction buffer (20 mM Tris, pH 7.5, 2 mM EDTA, 2 mM EGTA 50 mM β-glycerophosphate, 100 μM Na3 VO4 , 2 mM dithiothreitol [DTT], Complete protease inhibitor cocktail Roche) using approximately 0.5 mL of the extraction buffer for each 1 μg of plant material. .. In-gel kinase activity assays were performed using a method described previously (Wawer et al., ).

    Mass Spectrometry:

    Article Title: CenH3-Independent Kinetochore Assembly in Lepidoptera Requires CCAN, Including CENP-T.
    Article Snippet: .. Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres. .. Accurate chromosome segregation requires assembly of the multiprotein kinetochore complex at centromeres.

    Sonication:

    Article Title: mRNA Decapping and 5′-3′ Decay Contribute to the Regulation of ABA Signaling in Arabidopsis thaliana
    Article Snippet: .. In-gel kinase assay Frozen seedlings were ground in liquid nitrogen with mortar and pestle and sonicated three times for 20 s in the extraction buffer (20 mM Tris, pH 7.5, 2 mM EDTA, 2 mM EGTA 50 mM β-glycerophosphate, 100 μM Na3 VO4 , 2 mM dithiothreitol [DTT], Complete protease inhibitor cocktail Roche) using approximately 0.5 mL of the extraction buffer for each 1 μg of plant material. .. After sonication, the extracts were centrifuged at 18,000 rcf for 30 min at 4°C, and the supernatants were used for further studies.

    Lysis:

    Article Title: A single-dose plasmid-launched live-attenuated Zika vaccine induces protective immunity
    Article Snippet: .. Cells from the 6-well plates were washed once with PBS and lysed at 4 °C for 1 h in 200 μl RIPA lysis buffer (ThermoFisher Scientific) supplemented with 1× complete protease inhibitor cocktail (Roche). ..

    Article Title: The degradation of p53 and its major E3 ligase Mdm2 is differentially dependent on the proteasomal ubiquitin receptor S5a
    Article Snippet: Immunoprecipitation For proteasome immunoprecipitation, cells were washed twice in PBS and lysed in buffer 2: 20 mM Tris (pH 7.5), 0.1% NP-40, 5 mM MgCl2 , 10 mM NaF, 10 mM Na-pyrophosphate, 10 mM β-glycerophosphate, 20% glycerol, 2 mM ATP, 1 mM DTT, complete protease inhibitor cocktail Roche. .. Lysis was carried out on ice for 15 min with occasional vortexing.

    Article Title: A single-dose plasmid-launched live-attenuated Zika vaccine induces protective immunity
    Article Snippet: .. 2.6 SDS-PAGE and western blot Cells from the 6-well plates were washed once with PBS and lysed at 4 °C for 1 h in 200 μl RIPA lysis buffer (ThermoFisher Scientific) supplemented with 1× complete protease inhibitor cocktail (Roche). ..

    Kinase Assay:

    Article Title: mRNA Decapping and 5′-3′ Decay Contribute to the Regulation of ABA Signaling in Arabidopsis thaliana
    Article Snippet: .. In-gel kinase assay Frozen seedlings were ground in liquid nitrogen with mortar and pestle and sonicated three times for 20 s in the extraction buffer (20 mM Tris, pH 7.5, 2 mM EDTA, 2 mM EGTA 50 mM β-glycerophosphate, 100 μM Na3 VO4 , 2 mM dithiothreitol [DTT], Complete protease inhibitor cocktail Roche) using approximately 0.5 mL of the extraction buffer for each 1 μg of plant material. .. After sonication, the extracts were centrifuged at 18,000 rcf for 30 min at 4°C, and the supernatants were used for further studies.

    Western Blot:

    Article Title: Novel Potassium Channels in Kidney Mitochondria: The Hyperpolarization-Activated and Cyclic Nucleotide-Gated HCN Channels
    Article Snippet: Polyclonal voltage-dependent anion channel (VDAC, ab34726) was purchased from Abcam (Cambridge, UK) and Immobilon Western Chemiluminescent horseradish peroxidase (HRP) substrate was from Millipore (Mexico City, México). .. Anti-HCN1, anti-HCN2, anti-HCN3 and anti-HCN4 were purchased from Alomone (Jerusalem, Israel). cOmplete™ Protease Inhibitor Cocktail Roche, Mexico.

    Article Title: A single-dose plasmid-launched live-attenuated Zika vaccine induces protective immunity
    Article Snippet: Paragraph title: 2.6. SDS-PAGE and western blot ... Cells from the 6-well plates were washed once with PBS and lysed at 4 °C for 1 h in 200 μl RIPA lysis buffer (ThermoFisher Scientific) supplemented with 1× complete protease inhibitor cocktail (Roche).

    Article Title: A single-dose plasmid-launched live-attenuated Zika vaccine induces protective immunity
    Article Snippet: .. 2.6 SDS-PAGE and western blot Cells from the 6-well plates were washed once with PBS and lysed at 4 °C for 1 h in 200 μl RIPA lysis buffer (ThermoFisher Scientific) supplemented with 1× complete protease inhibitor cocktail (Roche). ..

    SDS Page:

    Article Title: mRNA Decapping and 5′-3′ Decay Contribute to the Regulation of ABA Signaling in Arabidopsis thaliana
    Article Snippet: In-gel kinase assay Frozen seedlings were ground in liquid nitrogen with mortar and pestle and sonicated three times for 20 s in the extraction buffer (20 mM Tris, pH 7.5, 2 mM EDTA, 2 mM EGTA 50 mM β-glycerophosphate, 100 μM Na3 VO4 , 2 mM dithiothreitol [DTT], Complete protease inhibitor cocktail Roche) using approximately 0.5 mL of the extraction buffer for each 1 μg of plant material. .. Briefly, protein samples were separated in 12% SDS/PAGE gels with 0.5 mg/ml histone embedded in the separating gel as a kinase substrate.

    Article Title: A single-dose plasmid-launched live-attenuated Zika vaccine induces protective immunity
    Article Snippet: Paragraph title: 2.6. SDS-PAGE and western blot ... Cells from the 6-well plates were washed once with PBS and lysed at 4 °C for 1 h in 200 μl RIPA lysis buffer (ThermoFisher Scientific) supplemented with 1× complete protease inhibitor cocktail (Roche).

    Article Title: A single-dose plasmid-launched live-attenuated Zika vaccine induces protective immunity
    Article Snippet: .. 2.6 SDS-PAGE and western blot Cells from the 6-well plates were washed once with PBS and lysed at 4 °C for 1 h in 200 μl RIPA lysis buffer (ThermoFisher Scientific) supplemented with 1× complete protease inhibitor cocktail (Roche). ..

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  • 99
    Roche complete mini protease inhibitor cocktail
    Expression of conventional breast cancer biomarkers and selected multidrug resistance genes. Cell lysates were prepared in radioimmunoprecipitation assay buffer supplemented with <t>cOmplete</t> Mini protease inhibitor and PhosSTOP phosphatase inhibitor. Quantities (10–50 μg) of total protein were run on a 10 % gel (MDR1), 4–20 % precast gels (epidermal growth factor receptor [EGFR], oestrogen receptor [ER], progesterone receptor [PR], type II topoisomerase [TOPO IIα]) and Any kD Mini-PROTEAN TGX Precast Protein Gels (human epidermal growth factor receptor 2 [HER2], HER3), transferred onto polyvinylidene fluoride membrane and developed using chemiluminescence substrate. Nat native, Epi-R epirubicin-resistant, GAPDH glyceraldehyde-3-phosphate dehydrogenase
    Complete Mini Protease Inhibitor Cocktail, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 350 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/complete mini protease inhibitor cocktail/product/Roche
    Average 99 stars, based on 350 article reviews
    Price from $9.99 to $1999.99
    complete mini protease inhibitor cocktail - by Bioz Stars, 2020-04
    99/100 stars
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    99
    Roche edta free protease inhibitor cocktail tablets
    Identification of the antigen recognized by mAb B4. A , Lysates prepared from isolated IEC were separated using a sequential three gel separation protocol as described in Materials and Methods . The presence of the B4 antigen was confirmed by Western blot analysis ( right ). Two gel samples ( gray color boxes ) were analyzed by MS. B , The MASCOT search results for the protein isolated from first sample was identified as mouse annexin IV based on the highest score number of 785; 78 matched peptides covering 53% of the annexin IV sequence were identified ( underlying bold letters ). C , Lysates of untransformed F-293 cells ( lane 1 ) and F-293 cells transformed with the pSecTag2/Hygro B expression vector carrying annexin IV insert (F-293-A4) ( lane 3 ) together with culture supernatants from transformed cells ( lane 2 ) were probed by Western blot analysis with mAb B4. D , Flow cytometric analysis of F-293 cells expressing recombinant annexin IV. F-293-A4 cells were probed in flow cytometry with mAb B4. E , Supernatant ( lane1 ) and lysate ( lane 2 ) from F-293-A4 cells expressing recombinant annexin IV that had been incubated in <t>PBS</t> alone, or supernatant ( lane 3 ) and lysate ( lane 4 ) from the same cells incubated with 0.5 M <t>EDTA,</t> were probed by Western blot analysis with mAb B4. Recombinant annexin IV was not released from F-293-A4 cells in the absence of free Ca 2+ .
    Edta Free Protease Inhibitor Cocktail Tablets, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 243 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche edta free complete protease inhibitor cocktail
    Polysomal phenotype of siRNA-mediated gene knock down of GYS1. (A) Quantitation of siRNA-mediated gene depletion of GYS1 and pGYS1. Extracts from HeLa cells, transfected with 50 nM GYS1 or control siRNAs, were separated by <t>SDS-polyacrylamide</t> gel electrophoresis and pGYS1 and GYS1 abundances examined by Western blot analysis. Actin was used as an internal control. (B) Polysomal distribution in control siRNA- and GYS1 siRNA-treated extracts. Cell lysates, treated as described in (2A) were separated by ultracentrifugation in 10–60% sucrose gradients. Overlays of the 254nm absorbance profiles are shown. (C) Polysome distribution in HeLa cells transfected with empty 3xFLAG vector or 3xFLAG-GYS1 DNA. Overlay of the 254nm absorbance profiles following separation of cells lysates in 10–60% sucrose gradients are shown. (D, E) Quantitation of ribosomal subunits in control siRNA- and GYS1 siRNA-treated extracts. Ribosomal subunits were released from polysomes and dissociated into 40S and 60S subunits by either treatment with 2 mM puromycin (D) or 15 mM <t>EDTA</t> (E). Cell lysates were separated in 10–60% sucrose gradient and absorbance was measured at 254 nm.
    Edta Free Complete Protease Inhibitor Cocktail, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 310 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Roche immunoprecipitation buffer
    The mutation of Leucine 49 into Glutamic acid (L49E) in the RRM of human RBPMS2 inhibits homodimerization, but does not alter binding to NOGGIN mRNA. ( A ) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by Duolink PLA in DF-1 cells that co-express Myc-RBPMS2 or Myc-RBPMS2-L49E and HA-RBPMS2, or Myc-RBPMS2 and HA-TC10. Ha-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Protein interactions were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm. ( B ) <t>Immunoprecipitation</t> of RBPMS2 homodimers. Protein lysates from DF-1 cells that express HA-RBPMS2 and Myc - RBPMS2 (lanes 2 and 3) or Myc-RBPMS2-L49E (lanes 4 and 5) were immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cellular extracts from cells that express HA-RBPMS2 alone. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). Immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( C ) Subcellular localization of human RBPMS2 and RBPMS2-L49E. HEK293 cells that express Myc-RBPMS2 or Myc-RBPMS2-L49E were detected with anti-EiF3n (eukaryotic translation initiation factor 3n is present in stress granule) and rabbit anti-Myc antibodies. Myc-RBPMS2 and Myc-RBPMS2-L49E show similar cytoplasmic localization. ( D ) Experimental small-angle X-ray scattering curve (logarithm of intensity in arbitrary units as a function of the momentum transfer range s in Å −1 ) for RBPMS2-Nter-L49E measured at 1.1 mg/ml (green crosses), with its fitting theoretical curve (red continuous line) back-calculated from the RBPMS2-Nter-L49E NMR structure (Supplementary Figure S2). Blue dots represent the relative error bound. The χ 2 value of the fit is 1.096. ( E ) EMSA binding assays using a fixed high concentration of 161-nt NOGGIN RNA (100 nM) were performed with increasing concentrations of RBPMS2-Nter and RBPMS2-Nter-L49E ranging from 0.1 to 5 μM on a same gel and detected with SYBR ® Green EMSA nucleic acid gel stain. Note that RBPMS2-Nter forms defined RNA/protein complex as soon as 1 μM and two complexes at 5 μM (complexes I and II), whereas RBPMS2-Nter-L49E forms very diffuse bands (bracket and arrow). Free 161-nt RNA is indicated with a red arrow.
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    Image Search Results


    Expression of conventional breast cancer biomarkers and selected multidrug resistance genes. Cell lysates were prepared in radioimmunoprecipitation assay buffer supplemented with cOmplete Mini protease inhibitor and PhosSTOP phosphatase inhibitor. Quantities (10–50 μg) of total protein were run on a 10 % gel (MDR1), 4–20 % precast gels (epidermal growth factor receptor [EGFR], oestrogen receptor [ER], progesterone receptor [PR], type II topoisomerase [TOPO IIα]) and Any kD Mini-PROTEAN TGX Precast Protein Gels (human epidermal growth factor receptor 2 [HER2], HER3), transferred onto polyvinylidene fluoride membrane and developed using chemiluminescence substrate. Nat native, Epi-R epirubicin-resistant, GAPDH glyceraldehyde-3-phosphate dehydrogenase

    Journal: Breast Cancer Research : BCR

    Article Title: Downregulation of histone H2A and H2B pathways is associated with anthracycline sensitivity in breast cancer

    doi: 10.1186/s13058-016-0676-6

    Figure Lengend Snippet: Expression of conventional breast cancer biomarkers and selected multidrug resistance genes. Cell lysates were prepared in radioimmunoprecipitation assay buffer supplemented with cOmplete Mini protease inhibitor and PhosSTOP phosphatase inhibitor. Quantities (10–50 μg) of total protein were run on a 10 % gel (MDR1), 4–20 % precast gels (epidermal growth factor receptor [EGFR], oestrogen receptor [ER], progesterone receptor [PR], type II topoisomerase [TOPO IIα]) and Any kD Mini-PROTEAN TGX Precast Protein Gels (human epidermal growth factor receptor 2 [HER2], HER3), transferred onto polyvinylidene fluoride membrane and developed using chemiluminescence substrate. Nat native, Epi-R epirubicin-resistant, GAPDH glyceraldehyde-3-phosphate dehydrogenase

    Article Snippet: WCL were prepared by adding equal volumes of 2× RIPA buffer supplemented with 25 U of Benzonase Nuclease (Sigma-Aldrich) and cOmplete Mini Protease Inhibitor Cocktail (Roche), incubating on ice for 30 minutes and sonicating for 15 minutes with 30-second on-off intervals.

    Techniques: Expressing, Radio Immunoprecipitation, Protease Inhibitor

    Identification of the antigen recognized by mAb B4. A , Lysates prepared from isolated IEC were separated using a sequential three gel separation protocol as described in Materials and Methods . The presence of the B4 antigen was confirmed by Western blot analysis ( right ). Two gel samples ( gray color boxes ) were analyzed by MS. B , The MASCOT search results for the protein isolated from first sample was identified as mouse annexin IV based on the highest score number of 785; 78 matched peptides covering 53% of the annexin IV sequence were identified ( underlying bold letters ). C , Lysates of untransformed F-293 cells ( lane 1 ) and F-293 cells transformed with the pSecTag2/Hygro B expression vector carrying annexin IV insert (F-293-A4) ( lane 3 ) together with culture supernatants from transformed cells ( lane 2 ) were probed by Western blot analysis with mAb B4. D , Flow cytometric analysis of F-293 cells expressing recombinant annexin IV. F-293-A4 cells were probed in flow cytometry with mAb B4. E , Supernatant ( lane1 ) and lysate ( lane 2 ) from F-293-A4 cells expressing recombinant annexin IV that had been incubated in PBS alone, or supernatant ( lane 3 ) and lysate ( lane 4 ) from the same cells incubated with 0.5 M EDTA, were probed by Western blot analysis with mAb B4. Recombinant annexin IV was not released from F-293-A4 cells in the absence of free Ca 2+ .

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: Pathogenic Natural Antibodies Recognizing Annexin IV Are Required to Develop Intestinal Ischemia-Reperfusion Injury

    doi: 10.4049/jimmunol.0803980

    Figure Lengend Snippet: Identification of the antigen recognized by mAb B4. A , Lysates prepared from isolated IEC were separated using a sequential three gel separation protocol as described in Materials and Methods . The presence of the B4 antigen was confirmed by Western blot analysis ( right ). Two gel samples ( gray color boxes ) were analyzed by MS. B , The MASCOT search results for the protein isolated from first sample was identified as mouse annexin IV based on the highest score number of 785; 78 matched peptides covering 53% of the annexin IV sequence were identified ( underlying bold letters ). C , Lysates of untransformed F-293 cells ( lane 1 ) and F-293 cells transformed with the pSecTag2/Hygro B expression vector carrying annexin IV insert (F-293-A4) ( lane 3 ) together with culture supernatants from transformed cells ( lane 2 ) were probed by Western blot analysis with mAb B4. D , Flow cytometric analysis of F-293 cells expressing recombinant annexin IV. F-293-A4 cells were probed in flow cytometry with mAb B4. E , Supernatant ( lane1 ) and lysate ( lane 2 ) from F-293-A4 cells expressing recombinant annexin IV that had been incubated in PBS alone, or supernatant ( lane 3 ) and lysate ( lane 4 ) from the same cells incubated with 0.5 M EDTA, were probed by Western blot analysis with mAb B4. Recombinant annexin IV was not released from F-293-A4 cells in the absence of free Ca 2+ .

    Article Snippet: After harvesting the cells, they were resuspended in PBS with Complete, EDTA-Free Protease Inhibitor Cocktail Tablets (Roche Molecular Biochemicals).

    Techniques: Isolation, Western Blot, Mass Spectrometry, Sequencing, Transformation Assay, Expressing, Plasmid Preparation, Flow Cytometry, Recombinant, Cytometry, Incubation

    Polysomal phenotype of siRNA-mediated gene knock down of GYS1. (A) Quantitation of siRNA-mediated gene depletion of GYS1 and pGYS1. Extracts from HeLa cells, transfected with 50 nM GYS1 or control siRNAs, were separated by SDS-polyacrylamide gel electrophoresis and pGYS1 and GYS1 abundances examined by Western blot analysis. Actin was used as an internal control. (B) Polysomal distribution in control siRNA- and GYS1 siRNA-treated extracts. Cell lysates, treated as described in (2A) were separated by ultracentrifugation in 10–60% sucrose gradients. Overlays of the 254nm absorbance profiles are shown. (C) Polysome distribution in HeLa cells transfected with empty 3xFLAG vector or 3xFLAG-GYS1 DNA. Overlay of the 254nm absorbance profiles following separation of cells lysates in 10–60% sucrose gradients are shown. (D, E) Quantitation of ribosomal subunits in control siRNA- and GYS1 siRNA-treated extracts. Ribosomal subunits were released from polysomes and dissociated into 40S and 60S subunits by either treatment with 2 mM puromycin (D) or 15 mM EDTA (E). Cell lysates were separated in 10–60% sucrose gradient and absorbance was measured at 254 nm.

    Journal: Journal of molecular biology

    Article Title: Proteomic Analysis of Ribosomes: Translational Control of mRNA populations by Glycogen Synthase GYS1

    doi: 10.1016/j.jmb.2011.04.064

    Figure Lengend Snippet: Polysomal phenotype of siRNA-mediated gene knock down of GYS1. (A) Quantitation of siRNA-mediated gene depletion of GYS1 and pGYS1. Extracts from HeLa cells, transfected with 50 nM GYS1 or control siRNAs, were separated by SDS-polyacrylamide gel electrophoresis and pGYS1 and GYS1 abundances examined by Western blot analysis. Actin was used as an internal control. (B) Polysomal distribution in control siRNA- and GYS1 siRNA-treated extracts. Cell lysates, treated as described in (2A) were separated by ultracentrifugation in 10–60% sucrose gradients. Overlays of the 254nm absorbance profiles are shown. (C) Polysome distribution in HeLa cells transfected with empty 3xFLAG vector or 3xFLAG-GYS1 DNA. Overlay of the 254nm absorbance profiles following separation of cells lysates in 10–60% sucrose gradients are shown. (D, E) Quantitation of ribosomal subunits in control siRNA- and GYS1 siRNA-treated extracts. Ribosomal subunits were released from polysomes and dissociated into 40S and 60S subunits by either treatment with 2 mM puromycin (D) or 15 mM EDTA (E). Cell lysates were separated in 10–60% sucrose gradient and absorbance was measured at 254 nm.

    Article Snippet: For preparation of total cell lysates, HeLa cells were washed and harvested in cold PBS, followed by lysis in RIPA buffer (150 mM NaCl, 1mM EDTA, 100 mM Tris-HCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing EDTA-free complete protease inhibitor cocktail (Roche).

    Techniques: Quantitation Assay, Transfection, Polyacrylamide Gel Electrophoresis, Western Blot, Plasmid Preparation

    The mutation of Leucine 49 into Glutamic acid (L49E) in the RRM of human RBPMS2 inhibits homodimerization, but does not alter binding to NOGGIN mRNA. ( A ) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by Duolink PLA in DF-1 cells that co-express Myc-RBPMS2 or Myc-RBPMS2-L49E and HA-RBPMS2, or Myc-RBPMS2 and HA-TC10. Ha-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Protein interactions were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm. ( B ) Immunoprecipitation of RBPMS2 homodimers. Protein lysates from DF-1 cells that express HA-RBPMS2 and Myc - RBPMS2 (lanes 2 and 3) or Myc-RBPMS2-L49E (lanes 4 and 5) were immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cellular extracts from cells that express HA-RBPMS2 alone. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). Immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( C ) Subcellular localization of human RBPMS2 and RBPMS2-L49E. HEK293 cells that express Myc-RBPMS2 or Myc-RBPMS2-L49E were detected with anti-EiF3n (eukaryotic translation initiation factor 3n is present in stress granule) and rabbit anti-Myc antibodies. Myc-RBPMS2 and Myc-RBPMS2-L49E show similar cytoplasmic localization. ( D ) Experimental small-angle X-ray scattering curve (logarithm of intensity in arbitrary units as a function of the momentum transfer range s in Å −1 ) for RBPMS2-Nter-L49E measured at 1.1 mg/ml (green crosses), with its fitting theoretical curve (red continuous line) back-calculated from the RBPMS2-Nter-L49E NMR structure (Supplementary Figure S2). Blue dots represent the relative error bound. The χ 2 value of the fit is 1.096. ( E ) EMSA binding assays using a fixed high concentration of 161-nt NOGGIN RNA (100 nM) were performed with increasing concentrations of RBPMS2-Nter and RBPMS2-Nter-L49E ranging from 0.1 to 5 μM on a same gel and detected with SYBR ® Green EMSA nucleic acid gel stain. Note that RBPMS2-Nter forms defined RNA/protein complex as soon as 1 μM and two complexes at 5 μM (complexes I and II), whereas RBPMS2-Nter-L49E forms very diffuse bands (bracket and arrow). Free 161-nt RNA is indicated with a red arrow.

    Journal: Nucleic Acids Research

    Article Title: Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity

    doi: 10.1093/nar/gku692

    Figure Lengend Snippet: The mutation of Leucine 49 into Glutamic acid (L49E) in the RRM of human RBPMS2 inhibits homodimerization, but does not alter binding to NOGGIN mRNA. ( A ) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by Duolink PLA in DF-1 cells that co-express Myc-RBPMS2 or Myc-RBPMS2-L49E and HA-RBPMS2, or Myc-RBPMS2 and HA-TC10. Ha-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Protein interactions were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm. ( B ) Immunoprecipitation of RBPMS2 homodimers. Protein lysates from DF-1 cells that express HA-RBPMS2 and Myc - RBPMS2 (lanes 2 and 3) or Myc-RBPMS2-L49E (lanes 4 and 5) were immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cellular extracts from cells that express HA-RBPMS2 alone. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). Immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( C ) Subcellular localization of human RBPMS2 and RBPMS2-L49E. HEK293 cells that express Myc-RBPMS2 or Myc-RBPMS2-L49E were detected with anti-EiF3n (eukaryotic translation initiation factor 3n is present in stress granule) and rabbit anti-Myc antibodies. Myc-RBPMS2 and Myc-RBPMS2-L49E show similar cytoplasmic localization. ( D ) Experimental small-angle X-ray scattering curve (logarithm of intensity in arbitrary units as a function of the momentum transfer range s in Å −1 ) for RBPMS2-Nter-L49E measured at 1.1 mg/ml (green crosses), with its fitting theoretical curve (red continuous line) back-calculated from the RBPMS2-Nter-L49E NMR structure (Supplementary Figure S2). Blue dots represent the relative error bound. The χ 2 value of the fit is 1.096. ( E ) EMSA binding assays using a fixed high concentration of 161-nt NOGGIN RNA (100 nM) were performed with increasing concentrations of RBPMS2-Nter and RBPMS2-Nter-L49E ranging from 0.1 to 5 μM on a same gel and detected with SYBR ® Green EMSA nucleic acid gel stain. Note that RBPMS2-Nter forms defined RNA/protein complex as soon as 1 μM and two complexes at 5 μM (complexes I and II), whereas RBPMS2-Nter-L49E forms very diffuse bands (bracket and arrow). Free 161-nt RNA is indicated with a red arrow.

    Article Snippet: For immunoprecipitation assays, 50 μg of total protein lysates were incubated in immunoprecipitation buffer (50-mM Tris pH8, 150-mM NaCl, 0.4% NP40, cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche)) with rabbit anti-Myc antibodies (Ozyme) pre-adsorbed to protein A-Sepharose CL-4B (GE Healthcare) at 4°C for 1 h and washed extensively.

    Techniques: Mutagenesis, Binding Assay, Proximity Ligation Assay, Labeling, Confocal Microscopy, Immunoprecipitation, Nuclear Magnetic Resonance, Concentration Assay, SYBR Green Assay, Staining

    RBPMS2 dimers are formed in vitro and in vivo independently of RBPMS2 interaction with RNA. ( A ) Schematic representation of the different RBPMS2 clones isolated by Y2H screening with human RBPMS2 as bait. The RRM domain of RBPMS2 is between amino acids 32 and 105. ( B ) Immunoprecipitation with rabbit anti-Myc antibodies (lanes 3 and 6) or without (lanes 2 and 5) of protein lysates from DF-1 cells that express human HA-RBPMS2 or HA-TC10 and Myc-RBPMS2 or not. Lanes 1 and 4: 10% of total protein extracts from cells that express only HA-RBPMS2 or HA-TC10. Co-immunoprecipitation of HA-RBPMS2 was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The efficiency of immunoprecipitation was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( C ) Co-immunoprecipitation of HA-RBPMS2 and Myc-RBPMS2 dimers in the absence of RNA. Protein lysates from DF-1 cells that express HA-RBPMS2 alone or with Myc-RBPMS2 (lanes 2–5) were incubated with 50-μg/ml RNase A at room temperature for 30 min (lanes 4 and 5) or left untreated (lanes 1–3) and then immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cell extracts from cells that express only HA-RBPMS2. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( D ) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by proximity ligation assays (PLAs) in DF-1 cells that express Myc-RBPMS2 with HA-RBPMS2 or HA-TC10, or Myc-NICD and HA-RBPMS2, or Myc-RBPMS2 alone. HA-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Interactions between proteins were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm.

    Journal: Nucleic Acids Research

    Article Title: Homodimerization of RBPMS2 through a new RRM-interaction motif is necessary to control smooth muscle plasticity

    doi: 10.1093/nar/gku692

    Figure Lengend Snippet: RBPMS2 dimers are formed in vitro and in vivo independently of RBPMS2 interaction with RNA. ( A ) Schematic representation of the different RBPMS2 clones isolated by Y2H screening with human RBPMS2 as bait. The RRM domain of RBPMS2 is between amino acids 32 and 105. ( B ) Immunoprecipitation with rabbit anti-Myc antibodies (lanes 3 and 6) or without (lanes 2 and 5) of protein lysates from DF-1 cells that express human HA-RBPMS2 or HA-TC10 and Myc-RBPMS2 or not. Lanes 1 and 4: 10% of total protein extracts from cells that express only HA-RBPMS2 or HA-TC10. Co-immunoprecipitation of HA-RBPMS2 was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The efficiency of immunoprecipitation was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( C ) Co-immunoprecipitation of HA-RBPMS2 and Myc-RBPMS2 dimers in the absence of RNA. Protein lysates from DF-1 cells that express HA-RBPMS2 alone or with Myc-RBPMS2 (lanes 2–5) were incubated with 50-μg/ml RNase A at room temperature for 30 min (lanes 4 and 5) or left untreated (lanes 1–3) and then immunoprecipitated with rabbit anti-Myc antibodies (lanes 3 and 5) or without (lanes 2 and 4). Lane 1: 10% of total cell extracts from cells that express only HA-RBPMS2. Co-immunoprecipitation was monitored by immunoblotting with mouse anti-HA antibodies (upper panel). The immunoprecipitation efficiency was monitored by immunoblotting with rabbit anti-Myc antibodies (lower panel). ( D ) Analysis of the interaction of Myc-RBPMS2 with HA-RBPMS2 by proximity ligation assays (PLAs) in DF-1 cells that express Myc-RBPMS2 with HA-RBPMS2 or HA-TC10, or Myc-NICD and HA-RBPMS2, or Myc-RBPMS2 alone. HA-tagged proteins were detected with anti-mouse HA antibodies (in green) and Myc-tagged proteins with anti-rabbit Myc antibodies (in red). Interactions between proteins were detected with Duolink PLA labeled in magenta. Images were collected by confocal microscopy. Bars, 10 μm.

    Article Snippet: For immunoprecipitation assays, 50 μg of total protein lysates were incubated in immunoprecipitation buffer (50-mM Tris pH8, 150-mM NaCl, 0.4% NP40, cOmplete, EDTA-free Protease Inhibitor Cocktail (Roche)) with rabbit anti-Myc antibodies (Ozyme) pre-adsorbed to protein A-Sepharose CL-4B (GE Healthcare) at 4°C for 1 h and washed extensively.

    Techniques: In Vitro, In Vivo, Clone Assay, Isolation, Immunoprecipitation, Incubation, Ligation, Proximity Ligation Assay, Labeling, Confocal Microscopy