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Roche 1×polymerase chain reaction pcr buffer
1×Polymerase Chain Reaction Pcr Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Polymerase Chain Reaction:

Article Title: Fragile X CGG Repeat Variation in Tamil Nadu, South India: A Comparison of Radioactive and Methylation-Specific Polymerase Chain Reaction in CGG Repeat Sizing
Article Snippet: .. FMR1 CGG repeats were amplified with 6 pmol of each primer c and f (Fu et al. , ) in 15 μL reaction volume containing 1×polymerase chain reaction (PCR) buffer (including 3.5 mM MgCl2 ), 200 μM each of dATP, dCTP and dTTP, 50 μM dGTP, 150 μM 7-deaza-dGTP (Roche), 10% DMSO (Sigma), 0.5 U of KlenTaq (AB Peptides), 2 μCi α32 PdCTP (BRIT), and 100 ng of genomic DNA. .. A negative control (without template) and a positive control (known premutation) were included in every PCR setup.

Article Title: Germline Transmission of a Novel Rat Embryonic Stem Cell Line Derived from Transgenic Rats
Article Snippet: .. RT-PCR was performed in 25 μL reactions containing 250 pg–250 ng cDNA, 1×polymerase chain reaction (PCR) buffer (Roche, Indianapolis, IN), 1.5 mM MgCl2 , 0.2 mM dNTPs, 0.2 μM of each primer, and 2.5 U of Roche FastStart Taq polymerase. ..

Reverse Transcription Polymerase Chain Reaction:

Article Title: Germline Transmission of a Novel Rat Embryonic Stem Cell Line Derived from Transgenic Rats
Article Snippet: .. RT-PCR was performed in 25 μL reactions containing 250 pg–250 ng cDNA, 1×polymerase chain reaction (PCR) buffer (Roche, Indianapolis, IN), 1.5 mM MgCl2 , 0.2 mM dNTPs, 0.2 μM of each primer, and 2.5 U of Roche FastStart Taq polymerase. ..

Amplification:

Article Title: Fragile X CGG Repeat Variation in Tamil Nadu, South India: A Comparison of Radioactive and Methylation-Specific Polymerase Chain Reaction in CGG Repeat Sizing
Article Snippet: .. FMR1 CGG repeats were amplified with 6 pmol of each primer c and f (Fu et al. , ) in 15 μL reaction volume containing 1×polymerase chain reaction (PCR) buffer (including 3.5 mM MgCl2 ), 200 μM each of dATP, dCTP and dTTP, 50 μM dGTP, 150 μM 7-deaza-dGTP (Roche), 10% DMSO (Sigma), 0.5 U of KlenTaq (AB Peptides), 2 μCi α32 PdCTP (BRIT), and 100 ng of genomic DNA. .. A negative control (without template) and a positive control (known premutation) were included in every PCR setup.

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  • 94
    Roche pcr buffer
    The linear range of the real-time <t>PCR</t> method when detecting purified <t>DNA</t> from C. jejuni CCUG 11284 on the RotorGene 3000 (5 × 10 1 to 1 × 10 7 copies; □) and the ABI-PRISM 7700 (10 3 to 10 7 copies; ▴). A real-time PCR sample
    Pcr Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 905 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr buffer/product/Roche
    Average 94 stars, based on 905 article reviews
    Price from $9.99 to $1999.99
    pcr buffer - by Bioz Stars, 2020-07
    94/100 stars
      Buy from Supplier

    85
    Roche traf6 buffer
    <t>TRAF6</t> interacts with WT and mutant N-HTT in vitro and full-length proteins in vivo and accumulates in mutant N-HTT aggregates. a , HEK 293 cells were transfected with FLAG-TRAF6, and the huntingtin N-terminal fragment fused to GFP (N-HTT-GFP) with WT (Gln 21 , Q21) or mutated (Gln 60 , Q60 and Gln 150 , Q150) polyglutamine expansion. Lysates were immunoprecipitated ( IP ) with anti-FLAG beads, and bound proteins were revealed by immunoblot ( IB ) with anti-GFP, Q150 and anti-FLAG antibodies. Lysates were tested for the expression of TRAF6 and N-HTT proteins. Molecular mass markers (kDa) are indicated on the left. b , cells were transfected with FLAG-TRAF6 and N-HTT-GFP constructs as indicated. Lysates were immunoprecipitated with anti-GFP. Bound proteins and lysates were analyzed with anti-FLAG and anti-GFP antibodies. An asterisk represents N-HTT bands from previous development of the same gel. # represents an unspecific band. c , HEK 293 cell lysates were immunoprecipitated with anti-HTT or control IgG (as indicated), and bound endogenous proteins were revealed with anti-TRAF6 and anti-HTT antibodies. d , the cortex from WT and homozygous Hdh Q111 ( mut ) mice was dissected, lysed, and used for immunoprecipitation of endogenous TRAF6 and full-length HTT proteins. e , the parietal cortex from HD post-mortem brain was lysed and processed for co-immunoprecipitation of endogenous proteins as in d. f , HEK 293 cells were transfected as in a . TRAF6 was visualized by indirect immunofluorescence with anti-FLAG antibody ( red ). N-HTT was visible by GFP autofluorescence ( green ). Nuclei were visualized with DAPI ( blue ). g , cells were transfected with N-HTT-GFP constructs. Endogenous TRAF6 was stained with anti-TRAF6 antibody ( red ).
    Traf6 Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/traf6 buffer/product/Roche
    Average 85 stars, based on 1 article reviews
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    traf6 buffer - by Bioz Stars, 2020-07
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    The linear range of the real-time PCR method when detecting purified DNA from C. jejuni CCUG 11284 on the RotorGene 3000 (5 × 10 1 to 1 × 10 7 copies; □) and the ABI-PRISM 7700 (10 3 to 10 7 copies; ▴). A real-time PCR sample

    Journal: Applied and Environmental Microbiology

    Article Title: Enrichment Followed by Quantitative PCR both for Rapid Detection and as a Tool for Quantitative Risk Assessment of Food-Borne Thermotolerant Campylobacters

    doi: 10.1128/AEM.70.6.3588-3592.2004

    Figure Lengend Snippet: The linear range of the real-time PCR method when detecting purified DNA from C. jejuni CCUG 11284 on the RotorGene 3000 (5 × 10 1 to 1 × 10 7 copies; □) and the ABI-PRISM 7700 (10 3 to 10 7 copies; ▴). A real-time PCR sample

    Article Snippet: The 25-μl real-time PCR mixture contained 1× PCR buffer for Tth DNA polymerase (Roche A/S, Hvidovre, Denmark), 1 U of Tth DNA polymerase (Roche A/S), 0.4 mM deoxynucleoside triphosphate mixture (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom), 0.44 μM forward primer 5′ CTG CTT AAC ACA AGT TGA GTA GG 3′, 0.48 μM reverse primer 5′ TTC CTT AGG TAC CGT CAG AA 3′ (DNA Technology, Århus, Denmark; C. jejuni 16S rRNA; GenBank accession no. ), 2.5 mM MgCl2 (Applied Biosystems), 30 μg of bovine serum albumin (BSA) for chicken samples and 5 μg of BSA for pure DNA (Roche A/S), 20 nM target Campylobacter probe labeled with 6-carboxyfluorescein (FAM; reporter dye) and 6-carboxytetramethylrhodamine (TAMRA; quencher dye) (5′ FAM-TGT CAT CCT CCA CGC GGC GTT GCT GC-TAMRA 3′; DNA Technology), 50 nM IAC probe (5′ VIC-TTC ATG AGG ACA CCT GAG TTG A-TAMRA 3′; Applied Biosystems), 5 × 103 copies of IAC (124 bp), and 5 μl of DNA sample.

    Techniques: Real-time Polymerase Chain Reaction, Purification

    Concordance of differential gene expression between DNA array and quantitative real time-PCR Analysis. Fold change values for negative controls and differentially expressed genes were generated by comparing untreated to CB2-treated samples. In all cases,

    Journal: Infection and Immunity

    Article Title: Whole-Genome DNA Array Analysis of the Response of Borrelia burgdorferi to a Bactericidal Monoclonal Antibody

    doi: 10.1128/IAI.72.4.2035-2044.2004

    Figure Lengend Snippet: Concordance of differential gene expression between DNA array and quantitative real time-PCR Analysis. Fold change values for negative controls and differentially expressed genes were generated by comparing untreated to CB2-treated samples. In all cases,

    Article Snippet: Each 50-μl final volume reaction contained 1 μl of DNA template (100 ng/μl); 1× PCR buffer (10 mM Tris-HCl, 1.5 mM MgCl2 , 50 mM KCl [pH 8.3]); dATP, dCTP, dGTP, and dTTP (10 mM each); BB0147FWD and BB0147REV (1 μM each); and 2 U of Taq DNA polymerase (Roche, Indianapolis, Ind.).

    Techniques: Expressing, DNA Array, Real-time Polymerase Chain Reaction, Generated

    Single cell RT-PCR analysis reveals clusters of CW3-specific clones expressing β chains encoded by identical VDJ-β nucleotide sequences paired with different TCR-α chains. The cDNA from tubes containing single CW3-specific CD8 T cells sorted from PBL of M-2 and M-3 or M-33 was subjected to RT-PCR, and PCR products were sequenced and assigned a TCR sequence code (NS). The complete analysis is shown in Figs. S1, S2, and S3, available at http://www.jem.org/cgi/content/full/jem.20021945/DC1 . Represented here are those cells for which both a CW3-like Vβ10 sequence and a CW3-like Vα3, Vα4, or Vα8 sequence were amplified. The deduced aa sequences of the TCR junctions are shown. All TCR-α sequences incorporate the Jα35 sequence. Also shown are the number (#) of cells found for each αβ TCR clone and its corresponding percentage (%) within the αβ TCR repertoire defined here for each mouse. Each cluster of αβ TCR clones sharing an identical Vβ10 TCR nucleotide sequence is framed. Cells for which an in-frame (IF) or out of frame (OF) second TCR-α rearrangement was also amplified are indicated.

    Journal: The Journal of Experimental Medicine

    Article Title: T Cell Receptor Gene Rearrangement Lineage Analysis Reveals Clues for the Origin of Highly Restricted Antigen-specific Repertoires

    doi: 10.1084/jem.20021945

    Figure Lengend Snippet: Single cell RT-PCR analysis reveals clusters of CW3-specific clones expressing β chains encoded by identical VDJ-β nucleotide sequences paired with different TCR-α chains. The cDNA from tubes containing single CW3-specific CD8 T cells sorted from PBL of M-2 and M-3 or M-33 was subjected to RT-PCR, and PCR products were sequenced and assigned a TCR sequence code (NS). The complete analysis is shown in Figs. S1, S2, and S3, available at http://www.jem.org/cgi/content/full/jem.20021945/DC1 . Represented here are those cells for which both a CW3-like Vβ10 sequence and a CW3-like Vα3, Vα4, or Vα8 sequence were amplified. The deduced aa sequences of the TCR junctions are shown. All TCR-α sequences incorporate the Jα35 sequence. Also shown are the number (#) of cells found for each αβ TCR clone and its corresponding percentage (%) within the αβ TCR repertoire defined here for each mouse. Each cluster of αβ TCR clones sharing an identical Vβ10 TCR nucleotide sequence is framed. Cells for which an in-frame (IF) or out of frame (OF) second TCR-α rearrangement was also amplified are indicated.

    Article Snippet: Cells gated as pCW3Kd+ Vβ10+ CD8+ were sorted as single cells into tubes containing 20 μl 1× PCR buffer (Roche) and 4 μg/ml 16S rRNA (Roche).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Clone Assay, Expressing, Polymerase Chain Reaction, Sequencing, Amplification

    Repertoire analysis by single cell DNA-PCR for coamplification of CW3-specific TCR VDJ-β and VJ-α sequences together with DJ-β rearrangements as potential clonal markers. For each of three mice (M-41, M-42, and M-43), single pCW3Kd + Vβ10 + CD8 + splenocytes were sorted 2 wk after immunization with P815-CW3 tumor cells. The rearranged TCR-α and TCR-β nucleotide sequences were amplified by single cell DNA-PCR. PCR products from cells with successful amplifications for TCR-α and TCR-β rearrangements were sequenced to identify paired αβ TCRs. Other details are as described for Fig. 2 .

    Journal: The Journal of Experimental Medicine

    Article Title: T Cell Receptor Gene Rearrangement Lineage Analysis Reveals Clues for the Origin of Highly Restricted Antigen-specific Repertoires

    doi: 10.1084/jem.20021945

    Figure Lengend Snippet: Repertoire analysis by single cell DNA-PCR for coamplification of CW3-specific TCR VDJ-β and VJ-α sequences together with DJ-β rearrangements as potential clonal markers. For each of three mice (M-41, M-42, and M-43), single pCW3Kd + Vβ10 + CD8 + splenocytes were sorted 2 wk after immunization with P815-CW3 tumor cells. The rearranged TCR-α and TCR-β nucleotide sequences were amplified by single cell DNA-PCR. PCR products from cells with successful amplifications for TCR-α and TCR-β rearrangements were sequenced to identify paired αβ TCRs. Other details are as described for Fig. 2 .

    Article Snippet: Cells gated as pCW3Kd+ Vβ10+ CD8+ were sorted as single cells into tubes containing 20 μl 1× PCR buffer (Roche) and 4 μg/ml 16S rRNA (Roche).

    Techniques: Polymerase Chain Reaction, Mouse Assay, Amplification

    TRAF6 interacts with WT and mutant N-HTT in vitro and full-length proteins in vivo and accumulates in mutant N-HTT aggregates. a , HEK 293 cells were transfected with FLAG-TRAF6, and the huntingtin N-terminal fragment fused to GFP (N-HTT-GFP) with WT (Gln 21 , Q21) or mutated (Gln 60 , Q60 and Gln 150 , Q150) polyglutamine expansion. Lysates were immunoprecipitated ( IP ) with anti-FLAG beads, and bound proteins were revealed by immunoblot ( IB ) with anti-GFP, Q150 and anti-FLAG antibodies. Lysates were tested for the expression of TRAF6 and N-HTT proteins. Molecular mass markers (kDa) are indicated on the left. b , cells were transfected with FLAG-TRAF6 and N-HTT-GFP constructs as indicated. Lysates were immunoprecipitated with anti-GFP. Bound proteins and lysates were analyzed with anti-FLAG and anti-GFP antibodies. An asterisk represents N-HTT bands from previous development of the same gel. # represents an unspecific band. c , HEK 293 cell lysates were immunoprecipitated with anti-HTT or control IgG (as indicated), and bound endogenous proteins were revealed with anti-TRAF6 and anti-HTT antibodies. d , the cortex from WT and homozygous Hdh Q111 ( mut ) mice was dissected, lysed, and used for immunoprecipitation of endogenous TRAF6 and full-length HTT proteins. e , the parietal cortex from HD post-mortem brain was lysed and processed for co-immunoprecipitation of endogenous proteins as in d. f , HEK 293 cells were transfected as in a . TRAF6 was visualized by indirect immunofluorescence with anti-FLAG antibody ( red ). N-HTT was visible by GFP autofluorescence ( green ). Nuclei were visualized with DAPI ( blue ). g , cells were transfected with N-HTT-GFP constructs. Endogenous TRAF6 was stained with anti-TRAF6 antibody ( red ).

    Journal: The Journal of Biological Chemistry

    Article Title: Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) Associates with Huntingtin Protein and Promotes Its Atypical Ubiquitination to Enhance Aggregate Formation *

    doi: 10.1074/jbc.M110.187591

    Figure Lengend Snippet: TRAF6 interacts with WT and mutant N-HTT in vitro and full-length proteins in vivo and accumulates in mutant N-HTT aggregates. a , HEK 293 cells were transfected with FLAG-TRAF6, and the huntingtin N-terminal fragment fused to GFP (N-HTT-GFP) with WT (Gln 21 , Q21) or mutated (Gln 60 , Q60 and Gln 150 , Q150) polyglutamine expansion. Lysates were immunoprecipitated ( IP ) with anti-FLAG beads, and bound proteins were revealed by immunoblot ( IB ) with anti-GFP, Q150 and anti-FLAG antibodies. Lysates were tested for the expression of TRAF6 and N-HTT proteins. Molecular mass markers (kDa) are indicated on the left. b , cells were transfected with FLAG-TRAF6 and N-HTT-GFP constructs as indicated. Lysates were immunoprecipitated with anti-GFP. Bound proteins and lysates were analyzed with anti-FLAG and anti-GFP antibodies. An asterisk represents N-HTT bands from previous development of the same gel. # represents an unspecific band. c , HEK 293 cell lysates were immunoprecipitated with anti-HTT or control IgG (as indicated), and bound endogenous proteins were revealed with anti-TRAF6 and anti-HTT antibodies. d , the cortex from WT and homozygous Hdh Q111 ( mut ) mice was dissected, lysed, and used for immunoprecipitation of endogenous TRAF6 and full-length HTT proteins. e , the parietal cortex from HD post-mortem brain was lysed and processed for co-immunoprecipitation of endogenous proteins as in d. f , HEK 293 cells were transfected as in a . TRAF6 was visualized by indirect immunofluorescence with anti-FLAG antibody ( red ). N-HTT was visible by GFP autofluorescence ( green ). Nuclei were visualized with DAPI ( blue ). g , cells were transfected with N-HTT-GFP constructs. Endogenous TRAF6 was stained with anti-TRAF6 antibody ( red ).

    Article Snippet: Immunoprecipitation and Western Blot For co-immunoprecipitation experiments, cells were lysed in TRAF6 buffer (200 mm NaCl, 50 mm Tris, pH 7.5, 0.5% Nonidet P-40, 10% glycerol) supplemented with anti-protease mixture (Roche Applied Science) and 5 mm N -ethylmaleimide.

    Techniques: Mutagenesis, In Vitro, In Vivo, Transfection, Immunoprecipitation, Expressing, Construct, Mouse Assay, Immunofluorescence, Staining

    TRAF6 enhances aggregate formation. a , HEK 293 cells were transfected with N-HTT-GFP alone ( C , control), or with FLAG-TRAF6 (WT or DN), as indicated. Immunofluorescence was performed with anti-FLAG ( red ) antibody. Nuclei were visualized with DAPI ( blue ). GFP was followed by autoflorescence. Confocal images from two independent experiments were scored for percentage of cells with aggregates ( b ), aggregate average size ( c ), and N-HTT cytoplasmic diffuse staining ( d ). At least 200 cells per experimental condition were counted. Data were analyzed with ImageJ software. Statistical analysis was performed with a t test. NS , not significant. AU , arbitrary units.

    Journal: The Journal of Biological Chemistry

    Article Title: Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) Associates with Huntingtin Protein and Promotes Its Atypical Ubiquitination to Enhance Aggregate Formation *

    doi: 10.1074/jbc.M110.187591

    Figure Lengend Snippet: TRAF6 enhances aggregate formation. a , HEK 293 cells were transfected with N-HTT-GFP alone ( C , control), or with FLAG-TRAF6 (WT or DN), as indicated. Immunofluorescence was performed with anti-FLAG ( red ) antibody. Nuclei were visualized with DAPI ( blue ). GFP was followed by autoflorescence. Confocal images from two independent experiments were scored for percentage of cells with aggregates ( b ), aggregate average size ( c ), and N-HTT cytoplasmic diffuse staining ( d ). At least 200 cells per experimental condition were counted. Data were analyzed with ImageJ software. Statistical analysis was performed with a t test. NS , not significant. AU , arbitrary units.

    Article Snippet: Immunoprecipitation and Western Blot For co-immunoprecipitation experiments, cells were lysed in TRAF6 buffer (200 mm NaCl, 50 mm Tris, pH 7.5, 0.5% Nonidet P-40, 10% glycerol) supplemented with anti-protease mixture (Roche Applied Science) and 5 mm N -ethylmaleimide.

    Techniques: Transfection, Immunofluorescence, Staining, Software

    Atypical ubiquitination by TRAF6 is localized at the aggregates. a , HEK 293 cells were transfected with Gln 150 N-HTT-GFP with FLAG-TRAF6 and HA-ubiquitin ( Ub ) WT. Localization of ubiquitin ( blue ) and TRAF6 ( red ) at N-HTT aggregates were analyzed by double immunofluorescence coupled with GFP autofluorescence. b , cells were transfected with Gln 150 N-HTT-GFP, FLAG-TRAF6, and HA-ubiquitin mutants Lys 6 , Lys 27 , Lys 29 , and Lys 11 as indicated. Aggregates were analyzed by immunofluorescence as in a .

    Journal: The Journal of Biological Chemistry

    Article Title: Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) Associates with Huntingtin Protein and Promotes Its Atypical Ubiquitination to Enhance Aggregate Formation *

    doi: 10.1074/jbc.M110.187591

    Figure Lengend Snippet: Atypical ubiquitination by TRAF6 is localized at the aggregates. a , HEK 293 cells were transfected with Gln 150 N-HTT-GFP with FLAG-TRAF6 and HA-ubiquitin ( Ub ) WT. Localization of ubiquitin ( blue ) and TRAF6 ( red ) at N-HTT aggregates were analyzed by double immunofluorescence coupled with GFP autofluorescence. b , cells were transfected with Gln 150 N-HTT-GFP, FLAG-TRAF6, and HA-ubiquitin mutants Lys 6 , Lys 27 , Lys 29 , and Lys 11 as indicated. Aggregates were analyzed by immunofluorescence as in a .

    Article Snippet: Immunoprecipitation and Western Blot For co-immunoprecipitation experiments, cells were lysed in TRAF6 buffer (200 mm NaCl, 50 mm Tris, pH 7.5, 0.5% Nonidet P-40, 10% glycerol) supplemented with anti-protease mixture (Roche Applied Science) and 5 mm N -ethylmaleimide.

    Techniques: Transfection, Immunofluorescence

    TRAF6 enhances ubiquitination of WT and mutant N-HTT. For ubiquitination assay, HEK 293 cells were transfected with HA-ubiquitin and N-HTT-GFP Gln 21 (Q21), Gln 60 (Q60), and Gln 150 (Q150) constructs with WT or DN FLAG-TRAF6. Lysates were immunoprecipitated ( IP ) with anti-GFP antibody, and ubiquitinated N-HTT protein was revealed with anti-HA. Input lysates were analyzed with anti-HA, anti-GFP, and anti-FLAG antibodies. Molecular mass markers (kDa) are indicated on the left. HC , heavy chain; IB , immunoblot; Ub , ubiquitin.

    Journal: The Journal of Biological Chemistry

    Article Title: Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) Associates with Huntingtin Protein and Promotes Its Atypical Ubiquitination to Enhance Aggregate Formation *

    doi: 10.1074/jbc.M110.187591

    Figure Lengend Snippet: TRAF6 enhances ubiquitination of WT and mutant N-HTT. For ubiquitination assay, HEK 293 cells were transfected with HA-ubiquitin and N-HTT-GFP Gln 21 (Q21), Gln 60 (Q60), and Gln 150 (Q150) constructs with WT or DN FLAG-TRAF6. Lysates were immunoprecipitated ( IP ) with anti-GFP antibody, and ubiquitinated N-HTT protein was revealed with anti-HA. Input lysates were analyzed with anti-HA, anti-GFP, and anti-FLAG antibodies. Molecular mass markers (kDa) are indicated on the left. HC , heavy chain; IB , immunoblot; Ub , ubiquitin.

    Article Snippet: Immunoprecipitation and Western Blot For co-immunoprecipitation experiments, cells were lysed in TRAF6 buffer (200 mm NaCl, 50 mm Tris, pH 7.5, 0.5% Nonidet P-40, 10% glycerol) supplemented with anti-protease mixture (Roche Applied Science) and 5 mm N -ethylmaleimide.

    Techniques: Mutagenesis, Ubiquitin Assay, Transfection, Construct, Immunoprecipitation

    In HD post-mortem brain, TRAF6 expression is increased, and TRAF6 protein accumulates in the insoluble fraction. a , total RNA was extracted from parietal cortex of HD and control brains. TRAF6 mRNA was measured by quantitative real-time PCR relative to β-actin. b , total protein lysates from HD and controls were prepared, and endogenous TRAF6 expression was monitored with anti-TRAF6 antibody. Loading was normalized by protein quantification and verified with anti-β-actin antibody. Representative images from three independent experiments are shown. c , densitometric analysis of protein bands was performed using Adobe Photoshop CS3. Expression of endogenous TRAF6 in total lysates was normalized relative to β-actin. Statistical analysis was performed with a Student's t test. d , soluble and insoluble fractions from HD and control brains were prepared and analyzed for TRAF6 distribution. Lysates were normalized for total protein content. Anti-TRAF6 and actin antibodies were used. Images are representative of three independent experiments. e , quantification of TRAF6 expression in insoluble fraction was done as described in c .

    Journal: The Journal of Biological Chemistry

    Article Title: Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) Associates with Huntingtin Protein and Promotes Its Atypical Ubiquitination to Enhance Aggregate Formation *

    doi: 10.1074/jbc.M110.187591

    Figure Lengend Snippet: In HD post-mortem brain, TRAF6 expression is increased, and TRAF6 protein accumulates in the insoluble fraction. a , total RNA was extracted from parietal cortex of HD and control brains. TRAF6 mRNA was measured by quantitative real-time PCR relative to β-actin. b , total protein lysates from HD and controls were prepared, and endogenous TRAF6 expression was monitored with anti-TRAF6 antibody. Loading was normalized by protein quantification and verified with anti-β-actin antibody. Representative images from three independent experiments are shown. c , densitometric analysis of protein bands was performed using Adobe Photoshop CS3. Expression of endogenous TRAF6 in total lysates was normalized relative to β-actin. Statistical analysis was performed with a Student's t test. d , soluble and insoluble fractions from HD and control brains were prepared and analyzed for TRAF6 distribution. Lysates were normalized for total protein content. Anti-TRAF6 and actin antibodies were used. Images are representative of three independent experiments. e , quantification of TRAF6 expression in insoluble fraction was done as described in c .

    Article Snippet: Immunoprecipitation and Western Blot For co-immunoprecipitation experiments, cells were lysed in TRAF6 buffer (200 mm NaCl, 50 mm Tris, pH 7.5, 0.5% Nonidet P-40, 10% glycerol) supplemented with anti-protease mixture (Roche Applied Science) and 5 mm N -ethylmaleimide.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    TRAF6 promotes atypical ubiquitination of WT and mutant N-HTT. HA-ubiquitin ( Ub ) mutants were used in which only the indicated lysine is available for chain formation. WT and Lys 0 ubiquitin were included as controls. HEK 293 cells were transfected with HA-ubiquitin mutant (as indicated), FLAG-TRAF6, and N-HTT-GFP Gln 21 (Q21) ( a ), Gln 60 (Q60) ( b ), and Gln 150 (Q150) ( c ). Ubiquitination was monitored with anti-HA, anti-GFP, and anti-FLAG antibodies. IB , immunoblot; IP , immunoprecipitation; HC , heavy chain.

    Journal: The Journal of Biological Chemistry

    Article Title: Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) Associates with Huntingtin Protein and Promotes Its Atypical Ubiquitination to Enhance Aggregate Formation *

    doi: 10.1074/jbc.M110.187591

    Figure Lengend Snippet: TRAF6 promotes atypical ubiquitination of WT and mutant N-HTT. HA-ubiquitin ( Ub ) mutants were used in which only the indicated lysine is available for chain formation. WT and Lys 0 ubiquitin were included as controls. HEK 293 cells were transfected with HA-ubiquitin mutant (as indicated), FLAG-TRAF6, and N-HTT-GFP Gln 21 (Q21) ( a ), Gln 60 (Q60) ( b ), and Gln 150 (Q150) ( c ). Ubiquitination was monitored with anti-HA, anti-GFP, and anti-FLAG antibodies. IB , immunoblot; IP , immunoprecipitation; HC , heavy chain.

    Article Snippet: Immunoprecipitation and Western Blot For co-immunoprecipitation experiments, cells were lysed in TRAF6 buffer (200 mm NaCl, 50 mm Tris, pH 7.5, 0.5% Nonidet P-40, 10% glycerol) supplemented with anti-protease mixture (Roche Applied Science) and 5 mm N -ethylmaleimide.

    Techniques: Mutagenesis, Transfection, Immunoprecipitation