nonidet p 40  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94

    Structured Review

    Thermo Fisher nonidet p 40
    Isolation of transition zones. ( A ) Live cells expressing eYFP::FTZC at the TZ (green) were ( B ) treated with 1% Nonidet P-40 to extract the soluble material. ( C ) Washed cytoskeletons were sonicated to shear the TZs from other flagellar material. Tagged
    Nonidet P 40, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nonidet p 40/product/Thermo Fisher
    Average 94 stars, based on 200 article reviews
    Price from $9.99 to $1999.99
    nonidet p 40 - by Bioz Stars, 2020-11
    94/100 stars

    Images

    1) Product Images from "Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes"

    Article Title: Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1604258113

    Isolation of transition zones. ( A ) Live cells expressing eYFP::FTZC at the TZ (green) were ( B ) treated with 1% Nonidet P-40 to extract the soluble material. ( C ) Washed cytoskeletons were sonicated to shear the TZs from other flagellar material. Tagged
    Figure Legend Snippet: Isolation of transition zones. ( A ) Live cells expressing eYFP::FTZC at the TZ (green) were ( B ) treated with 1% Nonidet P-40 to extract the soluble material. ( C ) Washed cytoskeletons were sonicated to shear the TZs from other flagellar material. Tagged

    Techniques Used: Isolation, Expressing, Sonication

    2) Product Images from "Plasminogen Activator Inhibitor Type 1 Interacts with ?3 Subunit of Proteasome and Modulates Its Activity *"

    Article Title: Plasminogen Activator Inhibitor Type 1 Interacts with ?3 Subunit of Proteasome and Modulates Its Activity *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.173781

    Direct interaction of PAI-1 with proteasome. Panel A  shows restriction analysis of five pGAD10 plasmids isolated from  His + LacZ +  colonies after yeast two-hybrid system screening. The encoded cDNA sequence, cloned into an EcoRI site, corresponded to the entire α3 subunit of human proteasome. Direct binding of PAI-1 to the proteasome was then confirmed by immunoprecipitation of the  125 I- PAI-1-proteasome complexes ( panel B ). Immunoprecipitated complexes of  125 I-labeled PAI-1 with proteasome were separated by SDS-PAGE (10% gels) under reducing conditions. The  first  and  third lanes  show complexes of  125 I-labeled PAI-1 with vitronectin ( Vn ) and proteasome, respectively. The  second  and  fourth lanes  show corresponding control immunoprecipitates.  Panel C  shows direct interaction of PAI-1 with proteasome analyzed by solid phase binding assay. Increasing doses of PAI-1 were incubated for 1 h at 37 °C in wells coated with proteasome. The unbound PAI-1 was washed away, and the bound PAI-1 was evaluated by ELISA using antibodies to PAI-1. Nonspecific binding observed in the absence of proteasome was subtracted. Data represent the mean ± S.D. obtained during three separate experiments.  Panel D  shows pulldown experiments using the His-tagged α3 proteasome subunit immobilized on NHS-Sepharose beads. EA.hy926 cells, control, or stimulated either with TNF (5 ng/ml and 20 ng/ml) or LPS (5 μg/ml and 20 μg/ml) were used as a source of PAI-1. PAI-1 was identified by Western immunoblotting with monoclonal anti-PAI-1 antibodies. Inputs of PAI-1 and β actin are shown. Direct interaction of both proteins in endothelial cells was further demonstrated by coimmunoprecipitation ( Panel E ). EA.hy926 cells, control and stimulated with TNF (5 ng/ml and 20 ng/ml) or LPS (5 μg/ml and 20 μg/ml), were lysed with 1% Nonidet P-40 lysis buffer and precipitated with anti-PAI-1 ( left ) or anti-α3 proteasome subunit ( right ) antibodies. Next, protein-protein complexes were captured by 100 μl of protein A/G-agarose beads. Bound proteins were solubilized by SDS sample buffer and subjected to SDS-PAGE and Western immunoblotting ( IB ) analysis. Coimmunoprecipitated ( IP ) proteins were detected with specific anti-α3 or anti-PAI-1 monoclonal antibodies. Inputs of α3 subunit and PAI-1 as well as blotting with nonimmune IgG and anti-actin antibodies are shown as well.
    Figure Legend Snippet: Direct interaction of PAI-1 with proteasome. Panel A shows restriction analysis of five pGAD10 plasmids isolated from His + LacZ + colonies after yeast two-hybrid system screening. The encoded cDNA sequence, cloned into an EcoRI site, corresponded to the entire α3 subunit of human proteasome. Direct binding of PAI-1 to the proteasome was then confirmed by immunoprecipitation of the 125 I- PAI-1-proteasome complexes ( panel B ). Immunoprecipitated complexes of 125 I-labeled PAI-1 with proteasome were separated by SDS-PAGE (10% gels) under reducing conditions. The first and third lanes show complexes of 125 I-labeled PAI-1 with vitronectin ( Vn ) and proteasome, respectively. The second and fourth lanes show corresponding control immunoprecipitates. Panel C shows direct interaction of PAI-1 with proteasome analyzed by solid phase binding assay. Increasing doses of PAI-1 were incubated for 1 h at 37 °C in wells coated with proteasome. The unbound PAI-1 was washed away, and the bound PAI-1 was evaluated by ELISA using antibodies to PAI-1. Nonspecific binding observed in the absence of proteasome was subtracted. Data represent the mean ± S.D. obtained during three separate experiments. Panel D shows pulldown experiments using the His-tagged α3 proteasome subunit immobilized on NHS-Sepharose beads. EA.hy926 cells, control, or stimulated either with TNF (5 ng/ml and 20 ng/ml) or LPS (5 μg/ml and 20 μg/ml) were used as a source of PAI-1. PAI-1 was identified by Western immunoblotting with monoclonal anti-PAI-1 antibodies. Inputs of PAI-1 and β actin are shown. Direct interaction of both proteins in endothelial cells was further demonstrated by coimmunoprecipitation ( Panel E ). EA.hy926 cells, control and stimulated with TNF (5 ng/ml and 20 ng/ml) or LPS (5 μg/ml and 20 μg/ml), were lysed with 1% Nonidet P-40 lysis buffer and precipitated with anti-PAI-1 ( left ) or anti-α3 proteasome subunit ( right ) antibodies. Next, protein-protein complexes were captured by 100 μl of protein A/G-agarose beads. Bound proteins were solubilized by SDS sample buffer and subjected to SDS-PAGE and Western immunoblotting ( IB ) analysis. Coimmunoprecipitated ( IP ) proteins were detected with specific anti-α3 or anti-PAI-1 monoclonal antibodies. Inputs of α3 subunit and PAI-1 as well as blotting with nonimmune IgG and anti-actin antibodies are shown as well.

    Techniques Used: Isolation, Sequencing, Clone Assay, Binding Assay, Immunoprecipitation, Labeling, SDS Page, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot, Lysis

    Interaction of PAI-1 with 20 S but not 26 S proteasome.  HeLa cells, which do not express endogenous PAI-1, were transfected with control pCMV or pCMV-PAI-1 for 24 and 48 h, lysed with 1% Nonidet P-40 buffer, and used to precipitate PAI-1 with anti PAI-1 antibodies. Then immunoprecipitates ( IP ) were probed for the proteasome 20 S (α3 and β1) or 26 S subunits (S4). To confirm the presence of the PAI-1-proteasome subunit complex, PVDF membranes were stripped and reprobed with anti-PAI-1 antibodies ( panel A ). In  panel B  samples of entire cell extracts were analyzed by Western blotting ( IB ) using antibodies to different proteasome subunits, namely α3, β1, and S4, as well as to β actin. In addition, normal mouse IgG was used as a control. Data are representative of those obtained during three separate experiments.
    Figure Legend Snippet: Interaction of PAI-1 with 20 S but not 26 S proteasome. HeLa cells, which do not express endogenous PAI-1, were transfected with control pCMV or pCMV-PAI-1 for 24 and 48 h, lysed with 1% Nonidet P-40 buffer, and used to precipitate PAI-1 with anti PAI-1 antibodies. Then immunoprecipitates ( IP ) were probed for the proteasome 20 S (α3 and β1) or 26 S subunits (S4). To confirm the presence of the PAI-1-proteasome subunit complex, PVDF membranes were stripped and reprobed with anti-PAI-1 antibodies ( panel A ). In panel B samples of entire cell extracts were analyzed by Western blotting ( IB ) using antibodies to different proteasome subunits, namely α3, β1, and S4, as well as to β actin. In addition, normal mouse IgG was used as a control. Data are representative of those obtained during three separate experiments.

    Techniques Used: Transfection, Western Blot

    3) Product Images from "Monoclonal Antibodies to Distinct Sites on Herpes Simplex Virus (HSV) Glycoprotein D Block HSV Binding to HVEM"

    Article Title: Monoclonal Antibodies to Distinct Sites on Herpes Simplex Virus (HSV) Glycoprotein D Block HSV Binding to HVEM

    Journal: Journal of Virology

    doi:

    Neutralization of HSV-1 entry by anti-gD MAb IgG. Purified HSV-1 KOS(tk12) was incubated with twofold dilutions of the MAb IgGs HD1 (group Ia), DL11 (group Ib), DL6 (group II), DL2 (group VI), and 1D3 (group VII) or nonimmune mouse IgG for 1 h at 37°C. Confluent CHO(250-2) cell monolayers on 96-well plates were infected with virus-Ab mixtures (4 × 10 4  PFU per well) for 1 h at 4°C and then shifted to 37°C for 7 h to allow for virus entry. Nonidet P-40 (0.1%) cell lysates were prepared, and then substrate was added, and β-galactosidase activity (milli-optical density units per minute) was read at 560 nm. One hundred percent entry corresponds to β-galactosidase activity in the absence of IgG. Each point represents the average of duplicate wells. Shown are the results of one representative experiment. The experiment was repeated three times with similar results.
    Figure Legend Snippet: Neutralization of HSV-1 entry by anti-gD MAb IgG. Purified HSV-1 KOS(tk12) was incubated with twofold dilutions of the MAb IgGs HD1 (group Ia), DL11 (group Ib), DL6 (group II), DL2 (group VI), and 1D3 (group VII) or nonimmune mouse IgG for 1 h at 37°C. Confluent CHO(250-2) cell monolayers on 96-well plates were infected with virus-Ab mixtures (4 × 10 4 PFU per well) for 1 h at 4°C and then shifted to 37°C for 7 h to allow for virus entry. Nonidet P-40 (0.1%) cell lysates were prepared, and then substrate was added, and β-galactosidase activity (milli-optical density units per minute) was read at 560 nm. One hundred percent entry corresponds to β-galactosidase activity in the absence of IgG. Each point represents the average of duplicate wells. Shown are the results of one representative experiment. The experiment was repeated three times with similar results.

    Techniques Used: Neutralization, Purification, Incubation, IA, Infection, Activity Assay

    4) Product Images from "Monoclonal Antibodies to Distinct Sites on Herpes Simplex Virus (HSV) Glycoprotein D Block HSV Binding to HVEM"

    Article Title: Monoclonal Antibodies to Distinct Sites on Herpes Simplex Virus (HSV) Glycoprotein D Block HSV Binding to HVEM

    Journal: Journal of Virology

    doi:

    Neutralization of HSV-1 entry by anti-gD MAb IgG. Purified HSV-1 KOS(tk12) was incubated with twofold dilutions of the MAb IgGs HD1 (group Ia), DL11 (group Ib), DL6 (group II), DL2 (group VI), and 1D3 (group VII) or nonimmune mouse IgG for 1 h at 37°C. Confluent CHO(250-2) cell monolayers on 96-well plates were infected with virus-Ab mixtures (4 × 10 4  PFU per well) for 1 h at 4°C and then shifted to 37°C for 7 h to allow for virus entry. Nonidet P-40 (0.1%) cell lysates were prepared, and then substrate was added, and β-galactosidase activity (milli-optical density units per minute) was read at 560 nm. One hundred percent entry corresponds to β-galactosidase activity in the absence of IgG. Each point represents the average of duplicate wells. Shown are the results of one representative experiment. The experiment was repeated three times with similar results.
    Figure Legend Snippet: Neutralization of HSV-1 entry by anti-gD MAb IgG. Purified HSV-1 KOS(tk12) was incubated with twofold dilutions of the MAb IgGs HD1 (group Ia), DL11 (group Ib), DL6 (group II), DL2 (group VI), and 1D3 (group VII) or nonimmune mouse IgG for 1 h at 37°C. Confluent CHO(250-2) cell monolayers on 96-well plates were infected with virus-Ab mixtures (4 × 10 4 PFU per well) for 1 h at 4°C and then shifted to 37°C for 7 h to allow for virus entry. Nonidet P-40 (0.1%) cell lysates were prepared, and then substrate was added, and β-galactosidase activity (milli-optical density units per minute) was read at 560 nm. One hundred percent entry corresponds to β-galactosidase activity in the absence of IgG. Each point represents the average of duplicate wells. Shown are the results of one representative experiment. The experiment was repeated three times with similar results.

    Techniques Used: Neutralization, Purification, Incubation, IA, Infection, Activity Assay

    5) Product Images from "A comparison of three approaches for the discovery of novel tripartite attachment complex proteins in Trypanosoma brucei"

    Article Title: A comparison of three approaches for the discovery of novel tripartite attachment complex proteins in Trypanosoma brucei

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0008568

    TAC102 enrichment and immunoprecipitation. The experiments were done in three independent replicates and all showed similar results. ( A ) Western blot analysis of the digitonin fractionation and subsequent lysis of the pellet fraction with 1% (v/v) Nonidet P-40 of whole cell protein from procyclic cells. T: total; S: supernatant; P: pellet. In orange: the pellet from the digitonin fractionation was used for the subsequent Nonidet P-40 lysis. The elongation factor EF1α served as a control. ( B ) Western blot analysis of the elutions (E1 and E2) of TAC102 immunoprecipitation replicates. ( C ) Silver stained SDS PAGE of TAC102 immunoprecipitation fractions. On the left are the molecular weights in kDa. Under each lane are written the equivalence of cells loaded. TAC102, indicated by a white arrow, was detected in E1. FT: flow-through; W1-W2-W3: washes; E1-E2: elutions. ( D ) Volcano plot representing the significance versus enrichment of the results from mass spectrometry analysis of TAC102 immunoprecipitation. A total of 775 proteins were identified from which 100 proteins were significantly enriched in the TAC102 immunoprecipitation (p ≤ 0.01). The two dotted lines represent the cut-off used (p ≤ 0.01 and enrichment  >  3). The protein the most enriched in the condition was TAC102. Three other TAC components were also highly enriched (p166, TAC40 and TAC60).
    Figure Legend Snippet: TAC102 enrichment and immunoprecipitation. The experiments were done in three independent replicates and all showed similar results. ( A ) Western blot analysis of the digitonin fractionation and subsequent lysis of the pellet fraction with 1% (v/v) Nonidet P-40 of whole cell protein from procyclic cells. T: total; S: supernatant; P: pellet. In orange: the pellet from the digitonin fractionation was used for the subsequent Nonidet P-40 lysis. The elongation factor EF1α served as a control. ( B ) Western blot analysis of the elutions (E1 and E2) of TAC102 immunoprecipitation replicates. ( C ) Silver stained SDS PAGE of TAC102 immunoprecipitation fractions. On the left are the molecular weights in kDa. Under each lane are written the equivalence of cells loaded. TAC102, indicated by a white arrow, was detected in E1. FT: flow-through; W1-W2-W3: washes; E1-E2: elutions. ( D ) Volcano plot representing the significance versus enrichment of the results from mass spectrometry analysis of TAC102 immunoprecipitation. A total of 775 proteins were identified from which 100 proteins were significantly enriched in the TAC102 immunoprecipitation (p ≤ 0.01). The two dotted lines represent the cut-off used (p ≤ 0.01 and enrichment > 3). The protein the most enriched in the condition was TAC102. Three other TAC components were also highly enriched (p166, TAC40 and TAC60).

    Techniques Used: Immunoprecipitation, Western Blot, Fractionation, Lysis, Staining, SDS Page, Mass Spectrometry

    6) Product Images from "Thrombin induces the release of the Y-box protein dbpB from mRNA: A mechanism of transcriptional activation"

    Article Title: Thrombin induces the release of the Y-box protein dbpB from mRNA: A mechanism of transcriptional activation

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.121592298

    ( A ) Latent dbpB is associated with RNA in nonstimulated EC. Cytosolic extracts were prepared from either control bovine aortic EC or EC treated with bovine α-thrombin (10 units/ml, 2 h), as described in  Experimental Procedures . Extracts from control EC were incubated with RNase A (1 mg/ml) or micrococcal nuclease (10,000 units/ml) for 30 min at 37°C before EMSA with the thrombin-response element (CCACCCACC) oligonucleotide as a probe. Arrow shows the position of dbpB activated by thrombin treatment of intact cells or ribonuclease treatment of extracts from untreated EC; McNase, micrococcal nuclease. ( B ) Latent dbpB coimmunoprecipitates with RNA. Cytosolic extracts prepared from control EC were incubated with preimmune serum or anti-dbpB antiserum raised against a C-terminal peptide of dbpB, followed by precipitation with protein A/G Sepharose. Protein was eluted by heat treatment. Aliquots of the eluants were treated with RNase A to release dbpB coimmunoprecipitated in association with RNA, and EMSA was performed. −Ab, preimmune serum used as a control; +Ab, immune serum used for precipitation of dbpB; RNase A, sample treated with ribonuclease A. ( C ) Oligo(dT) affinity chromatography of dbpB. ( Left ) EC cytosolic extracts were prepared from control and thrombin-treated EC as described in  Experimental Procedures  and dialyzed overnight in 0.2 M NaCl/0.01 M Tris⋅HCl, pH 7.5/0.002 M EDTA/0.2% Nonidet P-40/0.005 M β-mercaptoethanol. Oligo(dT)–biotin complex was added to the extract, and the mixture was applied to streptavidin magnetic beads. After rotation for 1 h at 4°C, the unbound fraction (fraction 1) was removed, the beads were washed three times, then washed with 25% formamide, and RNA was eluted with 60% formamide (fraction 2). The fractions were dialyzed overnight in EMSA buffer, and aliquots were treated with RNase A and subjected to EMSA. ( Right ) Fraction 1 from thrombin-treated EC lysates was used for EMSA without RNase treatment to detect activated dbpB.
    Figure Legend Snippet: ( A ) Latent dbpB is associated with RNA in nonstimulated EC. Cytosolic extracts were prepared from either control bovine aortic EC or EC treated with bovine α-thrombin (10 units/ml, 2 h), as described in Experimental Procedures . Extracts from control EC were incubated with RNase A (1 mg/ml) or micrococcal nuclease (10,000 units/ml) for 30 min at 37°C before EMSA with the thrombin-response element (CCACCCACC) oligonucleotide as a probe. Arrow shows the position of dbpB activated by thrombin treatment of intact cells or ribonuclease treatment of extracts from untreated EC; McNase, micrococcal nuclease. ( B ) Latent dbpB coimmunoprecipitates with RNA. Cytosolic extracts prepared from control EC were incubated with preimmune serum or anti-dbpB antiserum raised against a C-terminal peptide of dbpB, followed by precipitation with protein A/G Sepharose. Protein was eluted by heat treatment. Aliquots of the eluants were treated with RNase A to release dbpB coimmunoprecipitated in association with RNA, and EMSA was performed. −Ab, preimmune serum used as a control; +Ab, immune serum used for precipitation of dbpB; RNase A, sample treated with ribonuclease A. ( C ) Oligo(dT) affinity chromatography of dbpB. ( Left ) EC cytosolic extracts were prepared from control and thrombin-treated EC as described in Experimental Procedures and dialyzed overnight in 0.2 M NaCl/0.01 M Tris⋅HCl, pH 7.5/0.002 M EDTA/0.2% Nonidet P-40/0.005 M β-mercaptoethanol. Oligo(dT)–biotin complex was added to the extract, and the mixture was applied to streptavidin magnetic beads. After rotation for 1 h at 4°C, the unbound fraction (fraction 1) was removed, the beads were washed three times, then washed with 25% formamide, and RNA was eluted with 60% formamide (fraction 2). The fractions were dialyzed overnight in EMSA buffer, and aliquots were treated with RNase A and subjected to EMSA. ( Right ) Fraction 1 from thrombin-treated EC lysates was used for EMSA without RNase treatment to detect activated dbpB.

    Techniques Used: Incubation, Affinity Chromatography, Magnetic Beads

    7) Product Images from "The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination"

    Article Title: The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21176315

    Importance of ALG-2-binding to SARAF for Ca 2+ -dependent interaction between the SARAF CytDs. ( A ) Schematic diagram of expressed proteins and co-IP/HiBiT assay. ( B ) After HEK293 cells in 6-cm dishes had been co-transfected with expression plasmids for full-length HiBiTHA-SARAF (WT or F228S mutant) and for either StrepHA-SGFP2 (Control) or StrepHA-SARAF_CytD-SGFP2 (WT or F228S mutant) and cultured for 24 h, the cells were lysed with lysis buffer HKM containing 1% Nonidet P-40 supplemented with protease inhibitors, 10 mM NEM and 10 μM CaCl 2  ( Ca ) or 5 mM EGTA ( Eg ). SGFP2-fused proteins were immunoprecipitated with anti-GFP pAb, and the amounts of co-immunoprecipitated HiBiTHA-SARAF were estimated by the lytic HiBiT assay with LgBiT. The amounts of immunoprecipitated SGFP2-fused proteins were normalized by the StrepTactin-AP method as described in Materials and Methods. The relative amount of HiBiTHA-SARAF WT co-immunoprecipitated with StrepHA-SARAF_CytD-SGFP2 WT was expressed as 100% of relative interaction activity. The co-IP/HiBiT assay was performed in duplicate and repeated three times. Data are expressed as mean ± SEM ( n  = 3). Statistical significance by Tukey’s test is indicated in three different ways: asterisks for comparison between conditions of plus and minus Ca 2+ ;  a  for comparison with WT in the presence of Ca 2+ ;  b  for comparison with the control in the presence of Ca 2+ .  p  values were below 0.001 for all cases indicated (***,  a , and  b ). n.s., not significant. ( C ) Enhancement of the Ca 2+ -dependent CytD-to-CytD interaction of SARAF in HEK293 ALG-2KO cells by co-overexpression of FLAG-ALG-2. HEK293 ALG-2KO cells were co-transfected with expression plasmids for (i) full-length HiBiTHA-SARAF (WT), (ii) StrepHA-SARAF_CytD-SGFP2 (WT) and (iii) FLAG empty vector or FLAG-ALG-2 (WT, F85A or Y180A) and cultured for 24 h. The cells were lysed and subjected to HiBiT assay as described above. Relative interaction by co-overexpression of ALG-2 WT in the presence of Ca 2+  was expressed as 100% activity. Data are expressed as mean ± SEM ( n  = 3). Statistical significance by Tukey’s test is indicated by asterisks,  a , and  b  as described in ( B ) for comparison with the presence or absence of Ca 2+  (asterisks), WT ( a ) and control ( b ).
    Figure Legend Snippet: Importance of ALG-2-binding to SARAF for Ca 2+ -dependent interaction between the SARAF CytDs. ( A ) Schematic diagram of expressed proteins and co-IP/HiBiT assay. ( B ) After HEK293 cells in 6-cm dishes had been co-transfected with expression plasmids for full-length HiBiTHA-SARAF (WT or F228S mutant) and for either StrepHA-SGFP2 (Control) or StrepHA-SARAF_CytD-SGFP2 (WT or F228S mutant) and cultured for 24 h, the cells were lysed with lysis buffer HKM containing 1% Nonidet P-40 supplemented with protease inhibitors, 10 mM NEM and 10 μM CaCl 2 ( Ca ) or 5 mM EGTA ( Eg ). SGFP2-fused proteins were immunoprecipitated with anti-GFP pAb, and the amounts of co-immunoprecipitated HiBiTHA-SARAF were estimated by the lytic HiBiT assay with LgBiT. The amounts of immunoprecipitated SGFP2-fused proteins were normalized by the StrepTactin-AP method as described in Materials and Methods. The relative amount of HiBiTHA-SARAF WT co-immunoprecipitated with StrepHA-SARAF_CytD-SGFP2 WT was expressed as 100% of relative interaction activity. The co-IP/HiBiT assay was performed in duplicate and repeated three times. Data are expressed as mean ± SEM ( n = 3). Statistical significance by Tukey’s test is indicated in three different ways: asterisks for comparison between conditions of plus and minus Ca 2+ ; a for comparison with WT in the presence of Ca 2+ ; b for comparison with the control in the presence of Ca 2+ . p values were below 0.001 for all cases indicated (***, a , and b ). n.s., not significant. ( C ) Enhancement of the Ca 2+ -dependent CytD-to-CytD interaction of SARAF in HEK293 ALG-2KO cells by co-overexpression of FLAG-ALG-2. HEK293 ALG-2KO cells were co-transfected with expression plasmids for (i) full-length HiBiTHA-SARAF (WT), (ii) StrepHA-SARAF_CytD-SGFP2 (WT) and (iii) FLAG empty vector or FLAG-ALG-2 (WT, F85A or Y180A) and cultured for 24 h. The cells were lysed and subjected to HiBiT assay as described above. Relative interaction by co-overexpression of ALG-2 WT in the presence of Ca 2+ was expressed as 100% activity. Data are expressed as mean ± SEM ( n = 3). Statistical significance by Tukey’s test is indicated by asterisks, a , and b as described in ( B ) for comparison with the presence or absence of Ca 2+ (asterisks), WT ( a ) and control ( b ).

    Techniques Used: Binding Assay, Co-Immunoprecipitation Assay, Transfection, Expressing, Mutagenesis, Cell Culture, Lysis, Immunoprecipitation, Activity Assay, Over Expression, Plasmid Preparation

    Deletion mutation of the SARAF cytosolic domain (CytD) affecting ALG-2 binding and ubiquitination. ( A ) Schematic diagram of the SARAF CytD, which contains three distinct motifs rich in Pro and aromatic residues (designated regions 1, 2 and 3). Expression plasmids for the SARAF CytDs of WT and various deletion mutants that were fused with Strep-tag II and 3xHA (StrepHA) at the  N -terminus and SGFP2 at the C-terminus were constructed. ( B ) Strep-pulldown assay. After HEK293 cells had been transfected with pStrepHA-SARAF_CytD-SGFP2 (WT and deletion mutants) and cultured for 24 h, the cells were lysed with lysis buffer HKM containing protease inhibitors, 0.2% Nonidet P-40 and 10 μM CaCl 2 . The cleared cell lysates ( Input ) were subjected to Strep-pulldown followed by WB with anti-ALG-2 and anti-HA as indicated.
    Figure Legend Snippet: Deletion mutation of the SARAF cytosolic domain (CytD) affecting ALG-2 binding and ubiquitination. ( A ) Schematic diagram of the SARAF CytD, which contains three distinct motifs rich in Pro and aromatic residues (designated regions 1, 2 and 3). Expression plasmids for the SARAF CytDs of WT and various deletion mutants that were fused with Strep-tag II and 3xHA (StrepHA) at the N -terminus and SGFP2 at the C-terminus were constructed. ( B ) Strep-pulldown assay. After HEK293 cells had been transfected with pStrepHA-SARAF_CytD-SGFP2 (WT and deletion mutants) and cultured for 24 h, the cells were lysed with lysis buffer HKM containing protease inhibitors, 0.2% Nonidet P-40 and 10 μM CaCl 2 . The cleared cell lysates ( Input ) were subjected to Strep-pulldown followed by WB with anti-ALG-2 and anti-HA as indicated.

    Techniques Used: Mutagenesis, Binding Assay, Expressing, Strep-tag, Construct, Transfection, Cell Culture, Lysis, Western Blot

    Ubiquitination of SARAF by WWP1 ubiquitin E3 ligase. ( A ) Schematic diagrams of SGFP2-fused NEDD4 family ubiquitin E3 ligases investigated in this study. The E3 ligases contain the Ca 2+ -binding C2 domain, four repeats of WW domains, and the catalytic HECT domain. The catalytic Cys residues in each E3 ligases are shown in red lines. The active site Cys of WWP1 was substituted with Ala (C890A) and the HECT domain was deleted (ΔHECT). ( B ) Schematic diagrams representing StrepHA-SARAF mutants of Pro-to-Ala substitutions at PP X Y motifs and the CytD truncation. ( C ) StrepHA-SARAF was co-expressed in HEK293 cells with SGFP2-fused E3 ligase WWP1 (WT or C890A mutant), WWP2, ITCH or with a control vector ( Ctrl ). The cells were lysed in lysis buffer HK containing 1% Nonidet P-40 and E3 ligase inhibitors (2 mM EDTA, 10 mM NEM) as well as the protease inhibitor cocktail supplemented with 20 μM MG132. The cleared cell lysate ( Input ) was subjected to Strep-pulldown followed by WB with anti-HA and anti-GFP mAbs. Unmodified and ubiquitinated StrepHA-SARAF bands are marked with an arrowhead and unfilled arrowheads, respectively. ( D ) Strep-pulldown assays were performed using StrepHA-SARAF mutants and E3 ligase-defective mutants of SGFP2-WWP1 (C890A or ΔHECT) to determine important regions in SARAF for interaction with WWP1.
    Figure Legend Snippet: Ubiquitination of SARAF by WWP1 ubiquitin E3 ligase. ( A ) Schematic diagrams of SGFP2-fused NEDD4 family ubiquitin E3 ligases investigated in this study. The E3 ligases contain the Ca 2+ -binding C2 domain, four repeats of WW domains, and the catalytic HECT domain. The catalytic Cys residues in each E3 ligases are shown in red lines. The active site Cys of WWP1 was substituted with Ala (C890A) and the HECT domain was deleted (ΔHECT). ( B ) Schematic diagrams representing StrepHA-SARAF mutants of Pro-to-Ala substitutions at PP X Y motifs and the CytD truncation. ( C ) StrepHA-SARAF was co-expressed in HEK293 cells with SGFP2-fused E3 ligase WWP1 (WT or C890A mutant), WWP2, ITCH or with a control vector ( Ctrl ). The cells were lysed in lysis buffer HK containing 1% Nonidet P-40 and E3 ligase inhibitors (2 mM EDTA, 10 mM NEM) as well as the protease inhibitor cocktail supplemented with 20 μM MG132. The cleared cell lysate ( Input ) was subjected to Strep-pulldown followed by WB with anti-HA and anti-GFP mAbs. Unmodified and ubiquitinated StrepHA-SARAF bands are marked with an arrowhead and unfilled arrowheads, respectively. ( D ) Strep-pulldown assays were performed using StrepHA-SARAF mutants and E3 ligase-defective mutants of SGFP2-WWP1 (C890A or ΔHECT) to determine important regions in SARAF for interaction with WWP1.

    Techniques Used: Binding Assay, Mutagenesis, Plasmid Preparation, Lysis, Protease Inhibitor, Western Blot

    Related Articles

    Centrifugation:

    Article Title: Monoclonal Antibodies to Distinct Sites on Herpes Simplex Virus (HSV) Glycoprotein D Block HSV Binding to HVEM
    Article Snippet: .. Fifty-microliter reactions containing 3 μg of gD-1(Δ290–299t) and 16 μg of HVEMt per ml were incubated in binding buffer (10 mM Tris [pH 8.0], 100 mM NaCl, 0.1% Nonidet P-40, 0.05% bovine serum albumin [BSA], 0.05% chicken egg albumin) on ice for 1 h. MAb ascites (0.1 μl) was added for 1 h, followed by 50 μl of protein A-agarose (GIBCO BRL) (50 mg/ml) for 1 h. Bound material was collected by centrifugation at 13,000 × g for 3 min. Pellets were washed four times with high-salt buffer (10 mM Tris [pH 8.0], 500 mM NaCl, 0.1% Nonidet P-40, 0.05% BSA, 0.05% chicken egg albumin) and then boiled in SDS sample buffer for 3 min. .. Following SDS-PAGE (12% polyacrylamide), Western blots were probed with R7 and R140.

    BIA-KA:

    Article Title: Plasminogen Activator Inhibitor Type 1 Interacts with ?3 Subunit of Proteasome and Modulates Its Activity *
    Article Snippet: .. After incubation for 4 h, cells were collected and lysed with the Nonidet P-40 containing buffer, and cellular protein was concentration measured by the BCA method (Pierce/Thermo Scientific kit). ..

    Labeling:

    Article Title: Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes
    Article Snippet: .. Immunogold labeling of whole-mount cytoskeletons was performed by settling washed cells onto charged, formvar-coated grids, treating with 1% Nonidet P-40 in PBS for 5 min, block (1% BSA in PBS) for 20 min, staining with α-GFP (Invitrogen ; 1/500) in block for 45 min, washing three times in PBS, staining with 10 nm gold particle conjugated secondary antibody (Sigma G7402; 1/50) in block for 45 min, washing two times in PBS, followed by fixation for 10 min in 2.5% glutaraldehyde. .. Samples were then washed twice in PEME, stained for 1 s in 1% aurothioglucose, dried on filter paper, and imaged by TEM.

    Concentration Assay:

    Article Title: Plasminogen Activator Inhibitor Type 1 Interacts with ?3 Subunit of Proteasome and Modulates Its Activity *
    Article Snippet: .. After incubation for 4 h, cells were collected and lysed with the Nonidet P-40 containing buffer, and cellular protein was concentration measured by the BCA method (Pierce/Thermo Scientific kit). ..

    Incubation:

    Article Title: Monoclonal Antibodies to Distinct Sites on Herpes Simplex Virus (HSV) Glycoprotein D Block HSV Binding to HVEM
    Article Snippet: .. Fifty-microliter reactions containing 3 μg of gD-1(Δ290–299t) and 16 μg of HVEMt per ml were incubated in binding buffer (10 mM Tris [pH 8.0], 100 mM NaCl, 0.1% Nonidet P-40, 0.05% bovine serum albumin [BSA], 0.05% chicken egg albumin) on ice for 1 h. MAb ascites (0.1 μl) was added for 1 h, followed by 50 μl of protein A-agarose (GIBCO BRL) (50 mg/ml) for 1 h. Bound material was collected by centrifugation at 13,000 × g for 3 min. Pellets were washed four times with high-salt buffer (10 mM Tris [pH 8.0], 500 mM NaCl, 0.1% Nonidet P-40, 0.05% BSA, 0.05% chicken egg albumin) and then boiled in SDS sample buffer for 3 min. .. Following SDS-PAGE (12% polyacrylamide), Western blots were probed with R7 and R140.

    Article Title: Plasminogen Activator Inhibitor Type 1 Interacts with ?3 Subunit of Proteasome and Modulates Its Activity *
    Article Snippet: .. After incubation for 4 h, cells were collected and lysed with the Nonidet P-40 containing buffer, and cellular protein was concentration measured by the BCA method (Pierce/Thermo Scientific kit). ..

    Article Title: The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination
    Article Snippet: .. For pulldown-binding assay, aliquots of the suspensions of beads were incubated with 0.2 mL of NlucALG-2 solution (300 ng/mL) in lysis buffer HKM containing 0.2% Nonidet P-40, 0.1% gelatin, protease inhibitors, and 100 μM CaCl2 for 2 h at 4 °C. .. After the beads had been collected with a magnetic stand and washed three times with 0.5 mL of lysis buffer HKM containing 0.2% Nonidet P-40 and 100 μM CaCl2 , the beads were suspended with 50 μL of passive lysis buffer (PLB) (Promega, Madison, WI, USA).

    Blocking Assay:

    Article Title: Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes
    Article Snippet: .. Immunogold labeling of whole-mount cytoskeletons was performed by settling washed cells onto charged, formvar-coated grids, treating with 1% Nonidet P-40 in PBS for 5 min, block (1% BSA in PBS) for 20 min, staining with α-GFP (Invitrogen ; 1/500) in block for 45 min, washing three times in PBS, staining with 10 nm gold particle conjugated secondary antibody (Sigma G7402; 1/50) in block for 45 min, washing two times in PBS, followed by fixation for 10 min in 2.5% glutaraldehyde. .. Samples were then washed twice in PEME, stained for 1 s in 1% aurothioglucose, dried on filter paper, and imaged by TEM.

    End Labeling:

    Article Title: Thrombin induces the release of the Y-box protein dbpB from mRNA: A mechanism of transcriptional activation
    Article Snippet: .. N2 H-D-Phe-Pro-Arg-chloromethylketone was from Bachem, micrococcal nuclease (nuclease S7) and T4 kinase for end labeling of oligonucleotides were from Roche Molecular Biochemicals, [γ-32 P]dATP and [α-32 P]dCTP were from NEN, RNasin ribonuclease inhibitor was from Promega, Nonidet P-40 was from Pierce, and Trizol reagent, Lipofectin reagent, and oligo(dT)12–18 were from Life Technologies (Grand Island, NY). ..

    Western Blot:

    Article Title: The Extract of D. dasycarpus Ameliorates Oxazolone-Induced Skin Damage in Mice by Anti-Inflammatory and Antioxidant Mechanisms
    Article Snippet: .. Western Blot Analysis Cells were lysed in PBS containing 1% nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 5 μg/mL aprotinin, 100 μg/mL phenylmethylsulfonyl fluoride, 1 μg/mL pepstatin A, and 1 mM ethylenediaminetetraacetic acid (EDTA) at 4 °C for 20 min. Total lysates were quantified using a microBCA kit (Thermo Fisher Scientific, Waltham, MA, USA). .. Proteins (10 μg) were resolved by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to a PVDF (poly(vinylidene fluoride)) membrane.

    Lysis:

    Article Title: A comparison of three approaches for the discovery of novel tripartite attachment complex proteins in Trypanosoma brucei
    Article Snippet: .. The mitochondrial enriched fraction was then resuspended in ice-cold IP lysis/wash buffer (25 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% (v/v) Nonidet P-40, 5% (v/v) glycerol) containing protease inhibitors. .. After centrifugation (13 000 g, 10 min, 4°C), the supernatant was kept and put on beads containing anti-TAC102 monoclonal antibodies or empty beads (for negative controls), for 1:35 h at room temperature with rotation.

    Article Title: The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination
    Article Snippet: .. For pulldown-binding assay, aliquots of the suspensions of beads were incubated with 0.2 mL of NlucALG-2 solution (300 ng/mL) in lysis buffer HKM containing 0.2% Nonidet P-40, 0.1% gelatin, protease inhibitors, and 100 μM CaCl2 for 2 h at 4 °C. .. After the beads had been collected with a magnetic stand and washed three times with 0.5 mL of lysis buffer HKM containing 0.2% Nonidet P-40 and 100 μM CaCl2 , the beads were suspended with 50 μL of passive lysis buffer (PLB) (Promega, Madison, WI, USA).

    Binding Assay:

    Article Title: Monoclonal Antibodies to Distinct Sites on Herpes Simplex Virus (HSV) Glycoprotein D Block HSV Binding to HVEM
    Article Snippet: .. Fifty-microliter reactions containing 3 μg of gD-1(Δ290–299t) and 16 μg of HVEMt per ml were incubated in binding buffer (10 mM Tris [pH 8.0], 100 mM NaCl, 0.1% Nonidet P-40, 0.05% bovine serum albumin [BSA], 0.05% chicken egg albumin) on ice for 1 h. MAb ascites (0.1 μl) was added for 1 h, followed by 50 μl of protein A-agarose (GIBCO BRL) (50 mg/ml) for 1 h. Bound material was collected by centrifugation at 13,000 × g for 3 min. Pellets were washed four times with high-salt buffer (10 mM Tris [pH 8.0], 500 mM NaCl, 0.1% Nonidet P-40, 0.05% BSA, 0.05% chicken egg albumin) and then boiled in SDS sample buffer for 3 min. .. Following SDS-PAGE (12% polyacrylamide), Western blots were probed with R7 and R140.

    Staining:

    Article Title: Cilium transition zone proteome reveals compartmentalization and differential dynamics of ciliopathy complexes
    Article Snippet: .. Immunogold labeling of whole-mount cytoskeletons was performed by settling washed cells onto charged, formvar-coated grids, treating with 1% Nonidet P-40 in PBS for 5 min, block (1% BSA in PBS) for 20 min, staining with α-GFP (Invitrogen ; 1/500) in block for 45 min, washing three times in PBS, staining with 10 nm gold particle conjugated secondary antibody (Sigma G7402; 1/50) in block for 45 min, washing two times in PBS, followed by fixation for 10 min in 2.5% glutaraldehyde. .. Samples were then washed twice in PEME, stained for 1 s in 1% aurothioglucose, dried on filter paper, and imaged by TEM.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher rna processing buffer
    Dependence of <t>RNA</t> targeting on crRNA variants, temperature and point mutations a, <t>LbuC2c2</t> ssRNA target cleavage assay carried out, as per Methods with crRNAs possessing 16-nt, 20-nt or 24-nt spacers. b, LbuC2c2 ssRNA target cleavage time-course carried out at either 25°C and 37°C as per methods. c, LbuC2c2 ssRNA target cleavage timecourse carried out as per Methods with crRNAs possessing different 5′-flanking nucleotide mutations. Mutations are highlighted in red. 1–2 nucleotide 5′ extensions negligibly impacted cleavage efficiencies. In contrast, shortening the flanking region to 3 nts slowed cleavage rates. d Impact of point mutations on ribonuclease activity of C2c2 in conserved residue mutants within HEPN motifs for ssRNA targeting.
    Rna Processing Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna processing buffer/product/Thermo Fisher
    Average 99 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    rna processing buffer - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher emsa buffer
    Optimization of fluorescence labeling of MAGIC Factor and MAGIC Probes. ( A ) Labeling strategy of MAGIC Factor. MAGIC Factor was fluorescently labeled with increasing amounts of Alexa Fluor 488 SDP Ester and run on a 12% SDS-PAGE gel after purification by size-exclusion chromatography. The ratios refer to dye:protein molar ratios during fluorescent labeling. The control reaction was performed with DMSO instead of the dye. The same gel was imaged for native Alexa Fluor 488 fluorescence (top gel) and after staining all protein with Sypro Orange (bottom gel). ( B ) Electrophoretic Mobility Shift Assay <t>(EMSA)</t> of fluorescently labeled MAGIC Factor. Proteins from A were reacted with Alexa Fluor 647-labeled dsRNA and run on a native gel to visualize the binding affinities of the proteins. The gel was visualized in the <t>RNA</t> channel, protein channel and FRET channel. Note that the appearance of MAGIC Factor as multiple bands is likely due to the use of an NHS-ester dye to attach Alexa Fluor 488 to the protein. Dependent on the exact location of the fluorophore molecule, each single protein molecule likely runs differently on the native polyacrylamide gel. ( C ) Quantitative assessment of the EMSA with respect to the relative shift of the dsRNA, the corrected FRET (cFRET) intensity of shifted dsRNA and the cFRET/shift ratio. ( D ) Unlabeled and fluorescently labeled MAGIC Factor were affinity purified using dsRNA-coupled agarose beads. The proteins were reacted with the beads for 1 hr, washed three times with binding buffer and then gradually eluted with increasing concentrations of KCl. At the end, remaining proteins were eluted with 1x SDS-PAGE sample buffer and all samples run on a 12% SDS-PAGE gel. As a control experiment, fluorescently labeled MAGIC Factor was reacted with agarose beads in the absence of dsRNA. Gels were imaged for native Alexa Fluor 488 fluorescence (right gel) and after staining all protein (left gel). ( E ) Effect of degree of RNA fluorescent labeling on probe hybridization kinetics. Fluorescently labeled RNA probes were gel purified to obtain one to four labeled RNA probes. They were then reacted with unlabeled sense probes to generate dsRNA in TEN 100 buffer (tris, EDTA, sodium chloride) at 95°C (positive control) or physiologic buffer resembling cytoplasmic ionic concentrations at 37°C for 30 min or 2 hr. ssRNA are shown as controls. Representative native 20% polyacrylamide gels are shown. ( F ) Quantification of RNA fluorescence intensities from ( E ). Quantified data are shown as mean ± s.e.m. *p
    Emsa Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/emsa buffer/product/Thermo Fisher
    Average 99 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    emsa buffer - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher np 40 buffer
    DDC-induced ROS formation is associated with a depletion of energy metabolism and antioxidant enzymes and is regulated by GAPDH. (A) Hepatocytes from C3H and C57BL mice were left untreated (−) or treated with either 0.1% DMSO vehicle (0) or the indicated concentrations of DDC for 48 h. Equal protein amounts <t>(NP-40</t> lysates) were analyzed for expression of the indicated proteins. Samples from each strain were analyzed on separate gels. PDIA4 levels were unaltered (loading control). (B) C57BL hepatocytes were transfected with Control or GAPDH siRNA for 24 h and were then cultured for an additional 24 h in the presence of 0.025% DMSO vehicle (0) or 100 µM DDC or were left untreated (−). The NP-40 cell lysates were analyzed for the expression of the various proteins indicated. (C) Overexpression of Flag-tagged mouse GAPDH in isolated hepatocytes. NP-40 lysates were analyzed for expression of Flag-tagged GAPDH and total GAPDH. The Coomassie stain is included as a loading control. (D) Flag-GAPDH expression in C57BL hepatocytes, as determined by immunostaining with a mouse anti-Flag antibody (representing overexpressed GAPDH; green) and a rabbit anti-GAPDH antibody (representing total GAPDH; red). (E) C57BL hepatocytes were mock transfected (Control) or transfected with GAPDH siRNA or Flag-GAPDH mouse cDNA for 24 h and were then treated with 100 µM DDC for an additional 24 h. Representative images of ROS signal (green) with DAPI nuclear counterstain (blue) are shown. ROS levels (quantified as described in Materials and methods) exhibited a 2.2-fold increase after GAPDH knockdown and a 10-fold decrease after GAPDH overexpression relative to control. Bars, 20 µm.
    Np 40 Buffer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/np 40 buffer/product/Thermo Fisher
    Average 99 stars, based on 28 article reviews
    Price from $9.99 to $1999.99
    np 40 buffer - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    Image Search Results


    Dependence of RNA targeting on crRNA variants, temperature and point mutations a, LbuC2c2 ssRNA target cleavage assay carried out, as per Methods with crRNAs possessing 16-nt, 20-nt or 24-nt spacers. b, LbuC2c2 ssRNA target cleavage time-course carried out at either 25°C and 37°C as per methods. c, LbuC2c2 ssRNA target cleavage timecourse carried out as per Methods with crRNAs possessing different 5′-flanking nucleotide mutations. Mutations are highlighted in red. 1–2 nucleotide 5′ extensions negligibly impacted cleavage efficiencies. In contrast, shortening the flanking region to 3 nts slowed cleavage rates. d Impact of point mutations on ribonuclease activity of C2c2 in conserved residue mutants within HEPN motifs for ssRNA targeting.

    Journal: Nature

    Article Title: Two Distinct RNase Activities of CRISPR-C2c2 Enable Guide RNA Processing and RNA Detection

    doi: 10.1038/nature19802

    Figure Lengend Snippet: Dependence of RNA targeting on crRNA variants, temperature and point mutations a, LbuC2c2 ssRNA target cleavage assay carried out, as per Methods with crRNAs possessing 16-nt, 20-nt or 24-nt spacers. b, LbuC2c2 ssRNA target cleavage time-course carried out at either 25°C and 37°C as per methods. c, LbuC2c2 ssRNA target cleavage timecourse carried out as per Methods with crRNAs possessing different 5′-flanking nucleotide mutations. Mutations are highlighted in red. 1–2 nucleotide 5′ extensions negligibly impacted cleavage efficiencies. In contrast, shortening the flanking region to 3 nts slowed cleavage rates. d Impact of point mutations on ribonuclease activity of C2c2 in conserved residue mutants within HEPN motifs for ssRNA targeting.

    Article Snippet: These complexes were then diluted to 100nM LbuC2c2: 50 nM crRNA- λ 2 in RNA processing buffer (20 mM HEPES pH 6.8, 50 mM KCl, 5 mM MgCl2 , 10 μg/mL BSA, 10 μg/mL yeast tRNA, 0.01% Igepal CA-630 and 5% glycerol) in the presence of 185 nM of RNAase-Alert substrate (Thermo-Fisher), 100 ng of HeLa total RNA and increasing amounts of target 60 nt ssRNA (0–1 nM).

    Techniques: Cleavage Assay, Activity Assay

    Binding data for LbuC2c2 to mature crRNA and target ssRNA a, Filter binding assays were conducted as described in the Methods to determine the binding affinity of mature crRNA-A_GG to LbuC2c2-WT, LbuC2c2-dHEPN1, LbuC2c2-dHEPN2, or LbuC2c2-dHEPN1/dHEPN2. The quantified data were fit to standard binding isotherms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 27.1 ± 7.5 nM (LbuC2c2-WT), 15.2 ± 3.2 nM (LbuC2c2-dHEPN1), 11.5 ± 2.5 nM (LbuC2c2-dHEPN2), and 43.3 ± 11.5 nM (LbuC2c2- dHEPN1/dHEPN2). b, Representative electrophoretic mobility shift assay for binding reactions between LbuC2c2-dHEPN1/dHEPN2: crRNA-A_GG and either ‘on-target’ A ssRNA or ‘off-target’ B ssRNA, as indicated. Three independent experiments were conducted as described in the Methods. The gel was cropped for clarity. c, Quantified binding data from (b) were fitted to standard binding isoforms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 1.62 ± 0.43 nM for ssRNA A and N.D (≫10 nM) for ssRNA B. d, Filter binding assays were conducted as described in the Methods to determine the binding affinity of mature crRNA-A_GA to LbuC2c2-WT and LbuC2c2-R1079A. The quantified data were fit to standard binding isotherms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 4.65 ± 0.6 nM (LbuC2c2-WT) and 2.52 ± 0.5 nM (LbuC2c2-R1079A). It is of note that these binding affinities differ from panel a. This difference is accounted for in a slight difference in the 5′ sequence of the guide with panel a guides beginning with a 5′-G G CCA… and panel d 5′-G A CCA. While the native sequence guide (5′-G A CCA) binds tighter to LbuC2c2, no difference is seen in the RNA targeting efficiencies of these guide variants (). Extended Data Fig. 6c

    Journal: Nature

    Article Title: Two Distinct RNase Activities of CRISPR-C2c2 Enable Guide RNA Processing and RNA Detection

    doi: 10.1038/nature19802

    Figure Lengend Snippet: Binding data for LbuC2c2 to mature crRNA and target ssRNA a, Filter binding assays were conducted as described in the Methods to determine the binding affinity of mature crRNA-A_GG to LbuC2c2-WT, LbuC2c2-dHEPN1, LbuC2c2-dHEPN2, or LbuC2c2-dHEPN1/dHEPN2. The quantified data were fit to standard binding isotherms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 27.1 ± 7.5 nM (LbuC2c2-WT), 15.2 ± 3.2 nM (LbuC2c2-dHEPN1), 11.5 ± 2.5 nM (LbuC2c2-dHEPN2), and 43.3 ± 11.5 nM (LbuC2c2- dHEPN1/dHEPN2). b, Representative electrophoretic mobility shift assay for binding reactions between LbuC2c2-dHEPN1/dHEPN2: crRNA-A_GG and either ‘on-target’ A ssRNA or ‘off-target’ B ssRNA, as indicated. Three independent experiments were conducted as described in the Methods. The gel was cropped for clarity. c, Quantified binding data from (b) were fitted to standard binding isoforms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 1.62 ± 0.43 nM for ssRNA A and N.D (≫10 nM) for ssRNA B. d, Filter binding assays were conducted as described in the Methods to determine the binding affinity of mature crRNA-A_GA to LbuC2c2-WT and LbuC2c2-R1079A. The quantified data were fit to standard binding isotherms. Error bars represent the standard deviation from three independent experiments. Measured dissociation constants from three independent experiments (mean ± sd) were 4.65 ± 0.6 nM (LbuC2c2-WT) and 2.52 ± 0.5 nM (LbuC2c2-R1079A). It is of note that these binding affinities differ from panel a. This difference is accounted for in a slight difference in the 5′ sequence of the guide with panel a guides beginning with a 5′-G G CCA… and panel d 5′-G A CCA. While the native sequence guide (5′-G A CCA) binds tighter to LbuC2c2, no difference is seen in the RNA targeting efficiencies of these guide variants (). Extended Data Fig. 6c

    Article Snippet: These complexes were then diluted to 100nM LbuC2c2: 50 nM crRNA- λ 2 in RNA processing buffer (20 mM HEPES pH 6.8, 50 mM KCl, 5 mM MgCl2 , 10 μg/mL BSA, 10 μg/mL yeast tRNA, 0.01% Igepal CA-630 and 5% glycerol) in the presence of 185 nM of RNAase-Alert substrate (Thermo-Fisher), 100 ng of HeLa total RNA and increasing amounts of target 60 nt ssRNA (0–1 nM).

    Techniques: Binding Assay, Standard Deviation, Electrophoretic Mobility Shift Assay, Sequencing

    C2c2 provides sensitive detection of transcripts in complex mixtures a , Illustration of LbuC2c2 RNA detection approach using a quenched fluorescent RNA reporter. b , Quantification of fluorescence signal generated by LbuC2c2 after 30 min for varying concentrations of target RNA in the presence of human total RNA. RNase A shown as positive RNA degradation control. (mean ± s.d., n = 3) c ,. Quantification of fluorescence signal generated by LbuC2c2 loaded with a β -actin targeting crRNA after 3h for varying amounts of human total RNA or bacterial total RNA (as a β -actin null negative control). (mean ± s.d., n = 3) d , Tandem pre-crRNA processing also enables RNA detection. (mean ± s.d., n = 3) e , Model of the Type VI CRISPR pathway highlighting both of C2c2’s ribonuclease activities.

    Journal: Nature

    Article Title: Two Distinct RNase Activities of CRISPR-C2c2 Enable Guide RNA Processing and RNA Detection

    doi: 10.1038/nature19802

    Figure Lengend Snippet: C2c2 provides sensitive detection of transcripts in complex mixtures a , Illustration of LbuC2c2 RNA detection approach using a quenched fluorescent RNA reporter. b , Quantification of fluorescence signal generated by LbuC2c2 after 30 min for varying concentrations of target RNA in the presence of human total RNA. RNase A shown as positive RNA degradation control. (mean ± s.d., n = 3) c ,. Quantification of fluorescence signal generated by LbuC2c2 loaded with a β -actin targeting crRNA after 3h for varying amounts of human total RNA or bacterial total RNA (as a β -actin null negative control). (mean ± s.d., n = 3) d , Tandem pre-crRNA processing also enables RNA detection. (mean ± s.d., n = 3) e , Model of the Type VI CRISPR pathway highlighting both of C2c2’s ribonuclease activities.

    Article Snippet: These complexes were then diluted to 100nM LbuC2c2: 50 nM crRNA- λ 2 in RNA processing buffer (20 mM HEPES pH 6.8, 50 mM KCl, 5 mM MgCl2 , 10 μg/mL BSA, 10 μg/mL yeast tRNA, 0.01% Igepal CA-630 and 5% glycerol) in the presence of 185 nM of RNAase-Alert substrate (Thermo-Fisher), 100 ng of HeLa total RNA and increasing amounts of target 60 nt ssRNA (0–1 nM).

    Techniques: RNA Detection, Fluorescence, Generated, Negative Control, CRISPR

    Optimization of fluorescence labeling of MAGIC Factor and MAGIC Probes. ( A ) Labeling strategy of MAGIC Factor. MAGIC Factor was fluorescently labeled with increasing amounts of Alexa Fluor 488 SDP Ester and run on a 12% SDS-PAGE gel after purification by size-exclusion chromatography. The ratios refer to dye:protein molar ratios during fluorescent labeling. The control reaction was performed with DMSO instead of the dye. The same gel was imaged for native Alexa Fluor 488 fluorescence (top gel) and after staining all protein with Sypro Orange (bottom gel). ( B ) Electrophoretic Mobility Shift Assay (EMSA) of fluorescently labeled MAGIC Factor. Proteins from A were reacted with Alexa Fluor 647-labeled dsRNA and run on a native gel to visualize the binding affinities of the proteins. The gel was visualized in the RNA channel, protein channel and FRET channel. Note that the appearance of MAGIC Factor as multiple bands is likely due to the use of an NHS-ester dye to attach Alexa Fluor 488 to the protein. Dependent on the exact location of the fluorophore molecule, each single protein molecule likely runs differently on the native polyacrylamide gel. ( C ) Quantitative assessment of the EMSA with respect to the relative shift of the dsRNA, the corrected FRET (cFRET) intensity of shifted dsRNA and the cFRET/shift ratio. ( D ) Unlabeled and fluorescently labeled MAGIC Factor were affinity purified using dsRNA-coupled agarose beads. The proteins were reacted with the beads for 1 hr, washed three times with binding buffer and then gradually eluted with increasing concentrations of KCl. At the end, remaining proteins were eluted with 1x SDS-PAGE sample buffer and all samples run on a 12% SDS-PAGE gel. As a control experiment, fluorescently labeled MAGIC Factor was reacted with agarose beads in the absence of dsRNA. Gels were imaged for native Alexa Fluor 488 fluorescence (right gel) and after staining all protein (left gel). ( E ) Effect of degree of RNA fluorescent labeling on probe hybridization kinetics. Fluorescently labeled RNA probes were gel purified to obtain one to four labeled RNA probes. They were then reacted with unlabeled sense probes to generate dsRNA in TEN 100 buffer (tris, EDTA, sodium chloride) at 95°C (positive control) or physiologic buffer resembling cytoplasmic ionic concentrations at 37°C for 30 min or 2 hr. ssRNA are shown as controls. Representative native 20% polyacrylamide gels are shown. ( F ) Quantification of RNA fluorescence intensities from ( E ). Quantified data are shown as mean ± s.e.m. *p

    Journal: eLife

    Article Title: Multiplex live single-cell transcriptional analysis demarcates cellular functional heterogeneity

    doi: 10.7554/eLife.49599

    Figure Lengend Snippet: Optimization of fluorescence labeling of MAGIC Factor and MAGIC Probes. ( A ) Labeling strategy of MAGIC Factor. MAGIC Factor was fluorescently labeled with increasing amounts of Alexa Fluor 488 SDP Ester and run on a 12% SDS-PAGE gel after purification by size-exclusion chromatography. The ratios refer to dye:protein molar ratios during fluorescent labeling. The control reaction was performed with DMSO instead of the dye. The same gel was imaged for native Alexa Fluor 488 fluorescence (top gel) and after staining all protein with Sypro Orange (bottom gel). ( B ) Electrophoretic Mobility Shift Assay (EMSA) of fluorescently labeled MAGIC Factor. Proteins from A were reacted with Alexa Fluor 647-labeled dsRNA and run on a native gel to visualize the binding affinities of the proteins. The gel was visualized in the RNA channel, protein channel and FRET channel. Note that the appearance of MAGIC Factor as multiple bands is likely due to the use of an NHS-ester dye to attach Alexa Fluor 488 to the protein. Dependent on the exact location of the fluorophore molecule, each single protein molecule likely runs differently on the native polyacrylamide gel. ( C ) Quantitative assessment of the EMSA with respect to the relative shift of the dsRNA, the corrected FRET (cFRET) intensity of shifted dsRNA and the cFRET/shift ratio. ( D ) Unlabeled and fluorescently labeled MAGIC Factor were affinity purified using dsRNA-coupled agarose beads. The proteins were reacted with the beads for 1 hr, washed three times with binding buffer and then gradually eluted with increasing concentrations of KCl. At the end, remaining proteins were eluted with 1x SDS-PAGE sample buffer and all samples run on a 12% SDS-PAGE gel. As a control experiment, fluorescently labeled MAGIC Factor was reacted with agarose beads in the absence of dsRNA. Gels were imaged for native Alexa Fluor 488 fluorescence (right gel) and after staining all protein (left gel). ( E ) Effect of degree of RNA fluorescent labeling on probe hybridization kinetics. Fluorescently labeled RNA probes were gel purified to obtain one to four labeled RNA probes. They were then reacted with unlabeled sense probes to generate dsRNA in TEN 100 buffer (tris, EDTA, sodium chloride) at 95°C (positive control) or physiologic buffer resembling cytoplasmic ionic concentrations at 37°C for 30 min or 2 hr. ssRNA are shown as controls. Representative native 20% polyacrylamide gels are shown. ( F ) Quantification of RNA fluorescence intensities from ( E ). Quantified data are shown as mean ± s.e.m. *p

    Article Snippet: Typically, 100–200 nM of RNA was reacted with MAGIC Factor in EMSA buffer (25 mM HEPES pH 7.4, 100 mM KCl, 10 mM NaCl, 0.5 mM EDTA, 1 mM TCEP, 0.1% Nonidet P-40, 5% Glycerol), 10U Superase In RNase Inhibitor (Thermo Scientific) and 0.1 mg/ml t-RNA (Sigma Aldrich) for 30 min at 4°C before loading onto a 12% native polyacrylamide gel containing 2.5% glycerol.

    Techniques: Fluorescence, Labeling, SDS Page, Purification, Size-exclusion Chromatography, Staining, Electrophoretic Mobility Shift Assay, Binding Assay, Affinity Purification, Hybridization, Positive Control

    DDC-induced ROS formation is associated with a depletion of energy metabolism and antioxidant enzymes and is regulated by GAPDH. (A) Hepatocytes from C3H and C57BL mice were left untreated (−) or treated with either 0.1% DMSO vehicle (0) or the indicated concentrations of DDC for 48 h. Equal protein amounts (NP-40 lysates) were analyzed for expression of the indicated proteins. Samples from each strain were analyzed on separate gels. PDIA4 levels were unaltered (loading control). (B) C57BL hepatocytes were transfected with Control or GAPDH siRNA for 24 h and were then cultured for an additional 24 h in the presence of 0.025% DMSO vehicle (0) or 100 µM DDC or were left untreated (−). The NP-40 cell lysates were analyzed for the expression of the various proteins indicated. (C) Overexpression of Flag-tagged mouse GAPDH in isolated hepatocytes. NP-40 lysates were analyzed for expression of Flag-tagged GAPDH and total GAPDH. The Coomassie stain is included as a loading control. (D) Flag-GAPDH expression in C57BL hepatocytes, as determined by immunostaining with a mouse anti-Flag antibody (representing overexpressed GAPDH; green) and a rabbit anti-GAPDH antibody (representing total GAPDH; red). (E) C57BL hepatocytes were mock transfected (Control) or transfected with GAPDH siRNA or Flag-GAPDH mouse cDNA for 24 h and were then treated with 100 µM DDC for an additional 24 h. Representative images of ROS signal (green) with DAPI nuclear counterstain (blue) are shown. ROS levels (quantified as described in Materials and methods) exhibited a 2.2-fold increase after GAPDH knockdown and a 10-fold decrease after GAPDH overexpression relative to control. Bars, 20 µm.

    Journal: The Journal of Cell Biology

    Article Title: Energy determinants GAPDH and NDPK act as genetic modifiers for hepatocyte inclusion formation

    doi: 10.1083/jcb.201102142

    Figure Lengend Snippet: DDC-induced ROS formation is associated with a depletion of energy metabolism and antioxidant enzymes and is regulated by GAPDH. (A) Hepatocytes from C3H and C57BL mice were left untreated (−) or treated with either 0.1% DMSO vehicle (0) or the indicated concentrations of DDC for 48 h. Equal protein amounts (NP-40 lysates) were analyzed for expression of the indicated proteins. Samples from each strain were analyzed on separate gels. PDIA4 levels were unaltered (loading control). (B) C57BL hepatocytes were transfected with Control or GAPDH siRNA for 24 h and were then cultured for an additional 24 h in the presence of 0.025% DMSO vehicle (0) or 100 µM DDC or were left untreated (−). The NP-40 cell lysates were analyzed for the expression of the various proteins indicated. (C) Overexpression of Flag-tagged mouse GAPDH in isolated hepatocytes. NP-40 lysates were analyzed for expression of Flag-tagged GAPDH and total GAPDH. The Coomassie stain is included as a loading control. (D) Flag-GAPDH expression in C57BL hepatocytes, as determined by immunostaining with a mouse anti-Flag antibody (representing overexpressed GAPDH; green) and a rabbit anti-GAPDH antibody (representing total GAPDH; red). (E) C57BL hepatocytes were mock transfected (Control) or transfected with GAPDH siRNA or Flag-GAPDH mouse cDNA for 24 h and were then treated with 100 µM DDC for an additional 24 h. Representative images of ROS signal (green) with DAPI nuclear counterstain (blue) are shown. ROS levels (quantified as described in Materials and methods) exhibited a 2.2-fold increase after GAPDH knockdown and a 10-fold decrease after GAPDH overexpression relative to control. Bars, 20 µm.

    Article Snippet: Isolated hepatocytes were either lysed in NP-40 buffer or subjected to subcellular fractionation using the NE-PER cytoplasmic/nuclear fractionation kit (Thermo Fisher Scientific) to obtain total, cytoplasmic, and nuclear-enriched fractions.

    Techniques: Mouse Assay, Expressing, Transfection, Cell Culture, Over Expression, Isolation, Staining, Immunostaining