protein expression recombinant kh1  (Millipore)


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    Name:
    Proteins
    Description:
    This book provides an overview of the various medical diagnostic and industrial uses of proteins It has evolved from an earlier text Protein Biotechnology and includes the latest developments in the field and offers a varied selection of relevant case study material Initial chapters focus upon issues such as protein structure folding stability purification and characterization Later chapters detail the industrial production of proteins and their applications in medicine analysis and industry It presents a comprehensive review of proteins and enzymes used in the diagnostic therapeutic and industrial areas
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    p7492
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    Structured Review

    Millipore protein expression recombinant kh1
    Proteins
    This book provides an overview of the various medical diagnostic and industrial uses of proteins It has evolved from an earlier text Protein Biotechnology and includes the latest developments in the field and offers a varied selection of relevant case study material Initial chapters focus upon issues such as protein structure folding stability purification and characterization Later chapters detail the industrial production of proteins and their applications in medicine analysis and industry It presents a comprehensive review of proteins and enzymes used in the diagnostic therapeutic and industrial areas
    https://www.bioz.com/result/protein expression recombinant kh1/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    protein expression recombinant kh1 - by Bioz Stars, 2020-04
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    Images

    1) Product Images from "A method for the unbiased and efficient segmental labelling of RNA-binding proteins for structure and biophysics"

    Article Title: A method for the unbiased and efficient segmental labelling of RNA-binding proteins for structure and biophysics

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-13950-8

    KH1-KH23 ligation. ( a ) HPLC traces showing the formation of the MPAA thioester intermediate and ligated KH123 product (identified by cartoons) over time. Diphenyl column was used to resolve the KH23 and KH123 peaks. The unidentified ‘*’ impurity, which is at constant concentration during the reaction, provides a serendipitous control. Experiments were repeated three times. ( b ) (Left) Expected and measured masses of the reagents and product of the ligation. For the KH3 domain a 98% 15 N labelling has been assumed based on the level of enrichment of the 15 N nitrogen source. (Right) Reconstituted electrospray mass spectrum showing the de-convoluted molecular mass of the ligated KH123. ( c ) Superimposition of the backbone amide of 1 H{ 15 N} correlation experiments recorded on the KH3 and ligated KH3-only 15 N labelled KH123 proteins, indicating that the KH3 domain is correctly folded and assembled in the KH123 construct and highlighting the high quality of the NMR data. ( d ) The values of the 15 N longitudinal relaxation time of backbone amide groups in the ligated KH3 (black dots, this study) and of the isolated domains (orange dots, as previously reported 38 ) plotted along the protein sequence.
    Figure Legend Snippet: KH1-KH23 ligation. ( a ) HPLC traces showing the formation of the MPAA thioester intermediate and ligated KH123 product (identified by cartoons) over time. Diphenyl column was used to resolve the KH23 and KH123 peaks. The unidentified ‘*’ impurity, which is at constant concentration during the reaction, provides a serendipitous control. Experiments were repeated three times. ( b ) (Left) Expected and measured masses of the reagents and product of the ligation. For the KH3 domain a 98% 15 N labelling has been assumed based on the level of enrichment of the 15 N nitrogen source. (Right) Reconstituted electrospray mass spectrum showing the de-convoluted molecular mass of the ligated KH123. ( c ) Superimposition of the backbone amide of 1 H{ 15 N} correlation experiments recorded on the KH3 and ligated KH3-only 15 N labelled KH123 proteins, indicating that the KH3 domain is correctly folded and assembled in the KH123 construct and highlighting the high quality of the NMR data. ( d ) The values of the 15 N longitudinal relaxation time of backbone amide groups in the ligated KH3 (black dots, this study) and of the isolated domains (orange dots, as previously reported 38 ) plotted along the protein sequence.

    Techniques Used: Ligation, High Performance Liquid Chromatography, Concentration Assay, Construct, Nuclear Magnetic Resonance, Isolation, Sequencing

    Workflow of the expressed protein ligation protocol for the KH1, KH2, and KH3 RNA binding domains of the RNA regulator protein KSRP. The domains are expressed as either intein-CBD or SUMO-His fusion proteins and purified by chitin and nickel agarose affinity resins respectively. Intein fusion proteins are released from the chitin affinity resins by the strongly nucleophilic thiol additive benzyl mercaptan, which results in benzyl thioester formation at the C -terminus of the released protein. Instead, proteolytic cleavage catalysed by either SUMO or TEV proteases form N -terminal cysteine species. The ligations are performed at 40 °C in 6 M guanidine, pH 6.5 using TCEP as reducing agent and MPAA as a catalyst.
    Figure Legend Snippet: Workflow of the expressed protein ligation protocol for the KH1, KH2, and KH3 RNA binding domains of the RNA regulator protein KSRP. The domains are expressed as either intein-CBD or SUMO-His fusion proteins and purified by chitin and nickel agarose affinity resins respectively. Intein fusion proteins are released from the chitin affinity resins by the strongly nucleophilic thiol additive benzyl mercaptan, which results in benzyl thioester formation at the C -terminus of the released protein. Instead, proteolytic cleavage catalysed by either SUMO or TEV proteases form N -terminal cysteine species. The ligations are performed at 40 °C in 6 M guanidine, pH 6.5 using TCEP as reducing agent and MPAA as a catalyst.

    Techniques Used: Ligation, RNA Binding Assay, Purification

    Related Articles

    Centrifugation:

    Article Title: Deciphering fact from artifact when using reporter assays to investigate the roles of host factors on L1 retrotransposition
    Article Snippet: Protein expression ORF1p proteins were expressed in Rosetta (DE3) cells (Novagen) transformed with pET32aΔN-ORF1-His. .. Cultures were then induced with 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG), grown for an additional 4–6 h, pelleted via centrifugation and frozen at -80 °C.

    Expressing:

    Article Title: Deciphering fact from artifact when using reporter assays to investigate the roles of host factors on L1 retrotransposition
    Article Snippet: .. Protein expression ORF1p proteins were expressed in Rosetta (DE3) cells (Novagen) transformed with pET32aΔN-ORF1-His. ..

    Construct:

    Article Title: Factors influencing readthrough therapy for frequent cystic fibrosis premature termination codons
    Article Snippet: Purification and identification of readthrough proteins by mass spectrometry For readthrough GST purification, the yeast strain was transformed with pYX24-GST vectors and GST was purified as previously described [ ] unless the yeast was grown in the presence of 50 μg·mL−1 of paromomycin (Sigma Aldrich, St Louis, MO, USA). .. Proteins were identified with the Mascot algorithm and an in-house database constructed by merging SwissProt with user-generated GST sequences, each harbouring one of the 20 amino acids in the readthrough site.

    Electrophoresis:

    Article Title: A model for the molecular underpinnings of tooth defects in Axenfeld–Rieger syndrome
    Article Snippet: Following electrophoresis, the protein was transferred to polyvinylidene difluoride membrane filters (Millipore), immunoblotted and detected using specific antibodies and ECL reagents from Amersham Biosciences. .. The following polyclonal antibodies were used to detect the proteins: anti-β-tubulin (Santa Cruz Biotechnology), anti-Myc (Invitrogen) and Hmgn2 (Millipore).

    Incubation:

    Article Title: Characterization of DGCR8/Pasha, the essential cofactor for Drosha in primary miRNA processing
    Article Snippet: .. In vitro protein binding assay Immunopurified Drosha-FLAG proteins (∼2 µg) were immobilized on 15 µl of anti-FLAG M2 agarose mouse affinity gel (Sigma) and incubated with 106 c.p.m. of in vitro translated DGCR8 protein in 1 ml of buffer D-K′250 [20 mM Tris (pH 8.0), 250 mM KCl, 0.2 mM EDTA, 0.2 mM phenylmethlysulfonyl fluoride (PMSF), 0.1% Triton X-100]. .. After incubation for 90 min at 4°C, the resin was washed six times with 1 ml of buffer D-K′250 and the bound fraction was eluted by boiling in 40 µl of SDS–PAGE sample buffer and resolved by SDS–PAGE.

    Article Title: Sulfurtransferase and thioredoxin specifically interact as demonstrated by bimolecular fluorescence complementation analysis and biochemical tests
    Article Snippet: .. 5.8 Protein crosslinking Proteins (10 μM) were incubated at 25 °C with 1 mM bis(sulfosuccinimidyl) suberate (BS3 ) (Sigma) in 20 mM NaH2 PO4 , pH 7.0. .. At time intervals, aliquots were withdrawn from the reaction mixture and the crosslinking reactions were stopped in the SDS–PAGE loading buffer (125 mM Tris/HCl, pH 6.8, 4% SDS, 350 mM β-mercaptoethanol).

    Article Title: Binding of the Fkh1 Forkhead Associated Domain to a Phosphopeptide within the Mph1 DNA Helicase Regulates Mating-Type Switching in Budding Yeast
    Article Snippet: Lysates were then diluted 1:1 in CoIP buffer and incubated with the appropriate antibody. .. The starting extract and immunoprecipitated proteins were examined by protein immunoblotting using either anti-FLAG (ANTI-FLAG M2 monoclonal, Sigma) or anti-Fkh1 antibodies.

    Article Title: A model for the molecular underpinnings of tooth defects in Axenfeld–Rieger syndrome
    Article Snippet: .. Subsequently, sections were incubated with 10% goat serum-phosphate buffer saline-tween (PBST) for 30 min at room temperature, followed by overnight incubation at 4°C with an antibody (diluted 1:500) against one of the following proteins: Amel (Santa Cruz), Hmgn2 (Millipore), Ameb (Santa Cruz), Enml (Santa Cruz) or Dspp (Santa Cruz). .. After the incubation, the slides were treated with Alexa-488 (FITC channel)- or Alexa-555 (Cy3 channel)-labeled secondary antibody (Invitrogen) at a concentration of 1:500 for 30 min. Each antibody incubation was followed by three to six PBST (phosphate-buffered saline with 0.05% Tween-20) washes.

    Activity Assay:

    Article Title: The Pneumocystis Meiotic PCRan1p Kinase Exhibits Unique Temperature-Regulated Activity
    Article Snippet: To identify the native activity of P. carinii PCRan1p kinase within the organism, P. carinii cells were purified and protein extracts obtained as described above. .. The proteins were then immunoprecipitated using protein A-Sepharose beads (Sigma).

    Mass Spectrometry:

    Article Title: Factors influencing readthrough therapy for frequent cystic fibrosis premature termination codons
    Article Snippet: .. Purification and identification of readthrough proteins by mass spectrometry For readthrough GST purification, the yeast strain was transformed with pYX24-GST vectors and GST was purified as previously described [ ] unless the yeast was grown in the presence of 50 μg·mL−1 of paromomycin (Sigma Aldrich, St Louis, MO, USA). .. Mass spectrometry analysis was performed on Coomassie-stained gel bands corresponding to the readthrough GST proteins.

    Modification:

    Article Title: Binding of the Fkh1 Forkhead Associated Domain to a Phosphopeptide within the Mph1 DNA Helicase Regulates Mating-Type Switching in Budding Yeast
    Article Snippet: Co-immunoprecipitation of Fkh1 and FLAG-tagged Mph1 (modified from [ ]) were performed using Anti-FLAG antibodies (ANTI-FLAG M2 Affinity Gel, Sigma) or Protein A sepharose-linked anti-Fkh1 antibodies [ ]. .. The starting extract and immunoprecipitated proteins were examined by protein immunoblotting using either anti-FLAG (ANTI-FLAG M2 monoclonal, Sigma) or anti-Fkh1 antibodies.

    Article Title: O-GlcNAcylation of the Plum pox virus capsid protein catalyzed by SECRET AGENT: characterization of O-GlcNAc sites by electron transfer dissociation mass spectrometry
    Article Snippet: S-tagged PPV fusion proteins were detected using S-protein HRP conjugate (Novagen, Madison, WI) using the manufacturers protocol. .. To identify O -GlcNAc modified proteins, terminal GlcNAc moieties were labeled with [3 H]galactose using galactosyl transferase , with modifications described previously ( ; ).

    Western Blot:

    Article Title: Binding of the Fkh1 Forkhead Associated Domain to a Phosphopeptide within the Mph1 DNA Helicase Regulates Mating-Type Switching in Budding Yeast
    Article Snippet: Paragraph title: Co-immunoprecipitation and western blotting ... The starting extract and immunoprecipitated proteins were examined by protein immunoblotting using either anti-FLAG (ANTI-FLAG M2 monoclonal, Sigma) or anti-Fkh1 antibodies.

    Article Title: A model for the molecular underpinnings of tooth defects in Axenfeld–Rieger syndrome
    Article Snippet: Paragraph title: Western blot assays ... The following polyclonal antibodies were used to detect the proteins: anti-β-tubulin (Santa Cruz Biotechnology), anti-Myc (Invitrogen) and Hmgn2 (Millipore).

    Transformation Assay:

    Article Title: Deciphering fact from artifact when using reporter assays to investigate the roles of host factors on L1 retrotransposition
    Article Snippet: .. Protein expression ORF1p proteins were expressed in Rosetta (DE3) cells (Novagen) transformed with pET32aΔN-ORF1-His. ..

    Article Title: Factors influencing readthrough therapy for frequent cystic fibrosis premature termination codons
    Article Snippet: .. Purification and identification of readthrough proteins by mass spectrometry For readthrough GST purification, the yeast strain was transformed with pYX24-GST vectors and GST was purified as previously described [ ] unless the yeast was grown in the presence of 50 μg·mL−1 of paromomycin (Sigma Aldrich, St Louis, MO, USA). .. Mass spectrometry analysis was performed on Coomassie-stained gel bands corresponding to the readthrough GST proteins.

    Derivative Assay:

    Article Title: The Pneumocystis Meiotic PCRan1p Kinase Exhibits Unique Temperature-Regulated Activity
    Article Snippet: Paragraph title: Kinase Assays with In Vivo –Derived P. carinii PCRan1p ... The proteins were then immunoprecipitated using protein A-Sepharose beads (Sigma).

    Immunohistochemistry:

    Article Title: Genetic dissection of TAM receptor-ligand interaction in retinal pigment epithelial cell phagocytosis
    Article Snippet: .. We also tested the following Protein S antibodies for IHC: anti-protein S (AB15928; Millipore); anti-protein S (sc-25836; Santa Cruz); anti-protein S (AF4036; R & D Systems) and anti-protein S (P5180; Sigma). .. We used anti-opsin (MAB5356; Millipore) for the phagosome counts of .

    Article Title: A model for the molecular underpinnings of tooth defects in Axenfeld–Rieger syndrome
    Article Snippet: Paragraph title: Histology, fluorescent immunohistochemistry ... Subsequently, sections were incubated with 10% goat serum-phosphate buffer saline-tween (PBST) for 30 min at room temperature, followed by overnight incubation at 4°C with an antibody (diluted 1:500) against one of the following proteins: Amel (Santa Cruz), Hmgn2 (Millipore), Ameb (Santa Cruz), Enml (Santa Cruz) or Dspp (Santa Cruz).

    Immunoprecipitation:

    Article Title: The Pneumocystis Meiotic PCRan1p Kinase Exhibits Unique Temperature-Regulated Activity
    Article Snippet: .. The proteins were then immunoprecipitated using protein A-Sepharose beads (Sigma). .. The resulting proteins were then assayed for kinase activity as described above.

    Article Title: Binding of the Fkh1 Forkhead Associated Domain to a Phosphopeptide within the Mph1 DNA Helicase Regulates Mating-Type Switching in Budding Yeast
    Article Snippet: .. The starting extract and immunoprecipitated proteins were examined by protein immunoblotting using either anti-FLAG (ANTI-FLAG M2 monoclonal, Sigma) or anti-Fkh1 antibodies. ..

    Generated:

    Article Title: The Pneumocystis Meiotic PCRan1p Kinase Exhibits Unique Temperature-Regulated Activity
    Article Snippet: Subsequently, 100 μg of protein were precleared with a 1:250 dilution of PCRan1p preimmune sera, followed by a 1:250 dilution of a specific PCRan1p antibody generated previously ( ). .. The proteins were then immunoprecipitated using protein A-Sepharose beads (Sigma).

    other:

    Article Title:
    Article Snippet: It should be noted that we do not fully understand whether GluD1 and mGlu5 exhibit direct interaction, since endogenous proteins in HEK cells may coordinate an interaction between GluD1 and mGlu5.

    Article Title: Selective Actions of Novel Allosteric Modulators Reveal Functional Heteromers of Metabotropic Glutamate Receptors in the CNS
    Article Snippet: In cells coexpressing mGlu2 and mGlu4 , mGlu2 proteins were coprecipitated along with mGlu4 by mGlu4 antibody-coupled beads ( D ; ∼100 kDa and ∼240 kDa for monomeric and dimeric forms, respectively).

    Article Title: Abp1p and cortactin, new "hand-holds" for actin
    Article Snippet: Perhaps the best-characterized activators of the Arp2/3 complex are the Wiskott-Aldrich Syndrome protein (WASP) family proteins ( ).

    Article Title: The Pleckstrin Homology Domain-Containing Protein CKIP-1 Is Involved in Regulation of Cell Morphology and the Actin Cytoskeleton and Interaction with Actin Capping Protein
    Article Snippet: Here we provide evidence that CKIP-1 may serve to target CK2 to the plasma membrane and facilitate phosphorylation of the actin capping proteins by protein kinase CK2.

    Article Title: Hippocampal Homer1b/c is necessary for contextual fear conditioning and group I metabotropic glutamate receptor mediated long-term depression
    Article Snippet: These data are in agreement with previous findings which indicate that disruptions between mGluR5 and coiled coil Homer1 proteins impedes mGluR-LTD ( ).

    Imaging:

    Article Title: A model for the molecular underpinnings of tooth defects in Axenfeld–Rieger syndrome
    Article Snippet: Subsequently, sections were incubated with 10% goat serum-phosphate buffer saline-tween (PBST) for 30 min at room temperature, followed by overnight incubation at 4°C with an antibody (diluted 1:500) against one of the following proteins: Amel (Santa Cruz), Hmgn2 (Millipore), Ameb (Santa Cruz), Enml (Santa Cruz) or Dspp (Santa Cruz). .. Fluorescence signals from specific channels were quantified using imaging software (Nikon), and are presented as normalized mean intensity ± SEM.

    Nucleic Acid Electrophoresis:

    Article Title: A model for the molecular underpinnings of tooth defects in Axenfeld–Rieger syndrome
    Article Snippet: Approximately 25 μg of cell lysate was analyzed on sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels. .. The following polyclonal antibodies were used to detect the proteins: anti-β-tubulin (Santa Cruz Biotechnology), anti-Myc (Invitrogen) and Hmgn2 (Millipore).

    In Vivo:

    Article Title: The Pneumocystis Meiotic PCRan1p Kinase Exhibits Unique Temperature-Regulated Activity
    Article Snippet: Paragraph title: Kinase Assays with In Vivo –Derived P. carinii PCRan1p ... The proteins were then immunoprecipitated using protein A-Sepharose beads (Sigma).

    Fluorescence:

    Article Title: A model for the molecular underpinnings of tooth defects in Axenfeld–Rieger syndrome
    Article Snippet: Sections to be used for fluorescence immunohistochemistry were rehydrated and treated with 10 m m sodium citrate solution for 15 min at a slow boil for antigen retrieval. .. Subsequently, sections were incubated with 10% goat serum-phosphate buffer saline-tween (PBST) for 30 min at room temperature, followed by overnight incubation at 4°C with an antibody (diluted 1:500) against one of the following proteins: Amel (Santa Cruz), Hmgn2 (Millipore), Ameb (Santa Cruz), Enml (Santa Cruz) or Dspp (Santa Cruz).

    Labeling:

    Article Title: O-GlcNAcylation of the Plum pox virus capsid protein catalyzed by SECRET AGENT: characterization of O-GlcNAc sites by electron transfer dissociation mass spectrometry
    Article Snippet: S-tagged PPV fusion proteins were detected using S-protein HRP conjugate (Novagen, Madison, WI) using the manufacturers protocol. .. To identify O -GlcNAc modified proteins, terminal GlcNAc moieties were labeled with [3 H]galactose using galactosyl transferase , with modifications described previously ( ; ).

    Purification:

    Article Title: The Pneumocystis Meiotic PCRan1p Kinase Exhibits Unique Temperature-Regulated Activity
    Article Snippet: To identify the native activity of P. carinii PCRan1p kinase within the organism, P. carinii cells were purified and protein extracts obtained as described above. .. The proteins were then immunoprecipitated using protein A-Sepharose beads (Sigma).

    Article Title: Factors influencing readthrough therapy for frequent cystic fibrosis premature termination codons
    Article Snippet: .. Purification and identification of readthrough proteins by mass spectrometry For readthrough GST purification, the yeast strain was transformed with pYX24-GST vectors and GST was purified as previously described [ ] unless the yeast was grown in the presence of 50 μg·mL−1 of paromomycin (Sigma Aldrich, St Louis, MO, USA). .. Mass spectrometry analysis was performed on Coomassie-stained gel bands corresponding to the readthrough GST proteins.

    SDS Page:

    Article Title: Characterization of DGCR8/Pasha, the essential cofactor for Drosha in primary miRNA processing
    Article Snippet: In vitro protein binding assay Immunopurified Drosha-FLAG proteins (∼2 µg) were immobilized on 15 µl of anti-FLAG M2 agarose mouse affinity gel (Sigma) and incubated with 106 c.p.m. of in vitro translated DGCR8 protein in 1 ml of buffer D-K′250 [20 mM Tris (pH 8.0), 250 mM KCl, 0.2 mM EDTA, 0.2 mM phenylmethlysulfonyl fluoride (PMSF), 0.1% Triton X-100]. .. After incubation for 90 min at 4°C, the resin was washed six times with 1 ml of buffer D-K′250 and the bound fraction was eluted by boiling in 40 µl of SDS–PAGE sample buffer and resolved by SDS–PAGE.

    Article Title: Sulfurtransferase and thioredoxin specifically interact as demonstrated by bimolecular fluorescence complementation analysis and biochemical tests
    Article Snippet: 5.8 Protein crosslinking Proteins (10 μM) were incubated at 25 °C with 1 mM bis(sulfosuccinimidyl) suberate (BS3 ) (Sigma) in 20 mM NaH2 PO4 , pH 7.0. .. At time intervals, aliquots were withdrawn from the reaction mixture and the crosslinking reactions were stopped in the SDS–PAGE loading buffer (125 mM Tris/HCl, pH 6.8, 4% SDS, 350 mM β-mercaptoethanol).

    Article Title: O-GlcNAcylation of the Plum pox virus capsid protein catalyzed by SECRET AGENT: characterization of O-GlcNAc sites by electron transfer dissociation mass spectrometry
    Article Snippet: Proteins were separated by SDS-PAGE and transferred to Immobilon-P membrane (Millpore, Bedford, MA). .. S-tagged PPV fusion proteins were detected using S-protein HRP conjugate (Novagen, Madison, WI) using the manufacturers protocol.

    Plasmid Preparation:

    Article Title: A model for the molecular underpinnings of tooth defects in Axenfeld–Rieger syndrome
    Article Snippet: Subsequently, sections were incubated with 10% goat serum-phosphate buffer saline-tween (PBST) for 30 min at room temperature, followed by overnight incubation at 4°C with an antibody (diluted 1:500) against one of the following proteins: Amel (Santa Cruz), Hmgn2 (Millipore), Ameb (Santa Cruz), Enml (Santa Cruz) or Dspp (Santa Cruz). .. Nuclear counter staining was performed by applying 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting solution after the final wash (Vector Laboratories).

    Software:

    Article Title: A model for the molecular underpinnings of tooth defects in Axenfeld–Rieger syndrome
    Article Snippet: Subsequently, sections were incubated with 10% goat serum-phosphate buffer saline-tween (PBST) for 30 min at room temperature, followed by overnight incubation at 4°C with an antibody (diluted 1:500) against one of the following proteins: Amel (Santa Cruz), Hmgn2 (Millipore), Ameb (Santa Cruz), Enml (Santa Cruz) or Dspp (Santa Cruz). .. Fluorescence signals from specific channels were quantified using imaging software (Nikon), and are presented as normalized mean intensity ± SEM.

    Co-Immunoprecipitation Assay:

    Article Title: Binding of the Fkh1 Forkhead Associated Domain to a Phosphopeptide within the Mph1 DNA Helicase Regulates Mating-Type Switching in Budding Yeast
    Article Snippet: Beads were washed with CoIP buffer without detergents followed by washes with the same buffer with 200 mM NaCl. .. The starting extract and immunoprecipitated proteins were examined by protein immunoblotting using either anti-FLAG (ANTI-FLAG M2 monoclonal, Sigma) or anti-Fkh1 antibodies.

    In Vitro:

    Article Title: Characterization of DGCR8/Pasha, the essential cofactor for Drosha in primary miRNA processing
    Article Snippet: .. In vitro protein binding assay Immunopurified Drosha-FLAG proteins (∼2 µg) were immobilized on 15 µl of anti-FLAG M2 agarose mouse affinity gel (Sigma) and incubated with 106 c.p.m. of in vitro translated DGCR8 protein in 1 ml of buffer D-K′250 [20 mM Tris (pH 8.0), 250 mM KCl, 0.2 mM EDTA, 0.2 mM phenylmethlysulfonyl fluoride (PMSF), 0.1% Triton X-100]. .. After incubation for 90 min at 4°C, the resin was washed six times with 1 ml of buffer D-K′250 and the bound fraction was eluted by boiling in 40 µl of SDS–PAGE sample buffer and resolved by SDS–PAGE.

    Protein Binding:

    Article Title: Characterization of DGCR8/Pasha, the essential cofactor for Drosha in primary miRNA processing
    Article Snippet: .. In vitro protein binding assay Immunopurified Drosha-FLAG proteins (∼2 µg) were immobilized on 15 µl of anti-FLAG M2 agarose mouse affinity gel (Sigma) and incubated with 106 c.p.m. of in vitro translated DGCR8 protein in 1 ml of buffer D-K′250 [20 mM Tris (pH 8.0), 250 mM KCl, 0.2 mM EDTA, 0.2 mM phenylmethlysulfonyl fluoride (PMSF), 0.1% Triton X-100]. .. After incubation for 90 min at 4°C, the resin was washed six times with 1 ml of buffer D-K′250 and the bound fraction was eluted by boiling in 40 µl of SDS–PAGE sample buffer and resolved by SDS–PAGE.

    Concentration Assay:

    Article Title: A model for the molecular underpinnings of tooth defects in Axenfeld–Rieger syndrome
    Article Snippet: Subsequently, sections were incubated with 10% goat serum-phosphate buffer saline-tween (PBST) for 30 min at room temperature, followed by overnight incubation at 4°C with an antibody (diluted 1:500) against one of the following proteins: Amel (Santa Cruz), Hmgn2 (Millipore), Ameb (Santa Cruz), Enml (Santa Cruz) or Dspp (Santa Cruz). .. After the incubation, the slides were treated with Alexa-488 (FITC channel)- or Alexa-555 (Cy3 channel)-labeled secondary antibody (Invitrogen) at a concentration of 1:500 for 30 min. Each antibody incubation was followed by three to six PBST (phosphate-buffered saline with 0.05% Tween-20) washes.

    Staining:

    Article Title: Sulfurtransferase and thioredoxin specifically interact as demonstrated by bimolecular fluorescence complementation analysis and biochemical tests
    Article Snippet: 5.8 Protein crosslinking Proteins (10 μM) were incubated at 25 °C with 1 mM bis(sulfosuccinimidyl) suberate (BS3 ) (Sigma) in 20 mM NaH2 PO4 , pH 7.0. .. Samples were run on 15% SDS–PAGE gels and stained with blue silver (0.12% Coomassie Blue G-250, 10% ammonium sulfate, 10% phosphoric acid, 20% methanol) in order to detect the protein products.

    Article Title: A model for the molecular underpinnings of tooth defects in Axenfeld–Rieger syndrome
    Article Snippet: Sections were cut to 7 μm thickness, and for each sample multiple sections were stained by standard hematoxylin and eEosin staining to assess sample quality. .. Subsequently, sections were incubated with 10% goat serum-phosphate buffer saline-tween (PBST) for 30 min at room temperature, followed by overnight incubation at 4°C with an antibody (diluted 1:500) against one of the following proteins: Amel (Santa Cruz), Hmgn2 (Millipore), Ameb (Santa Cruz), Enml (Santa Cruz) or Dspp (Santa Cruz).

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  • 85
    Millipore nova 1 kh1 2
    ( A ) Nitrocellulose filter binding assays of polycytidylate (C 17 ) RNA against wild-type hnRNP E1 <t>KH1/2</t> protein and mutant protein harboring R(32)→K mutations in both KH domains. ( B ) against wild-type and Q(32)→R <t>Nova-1</t> KH3 proteins].
    Nova 1 Kh1 2, supplied by Millipore, 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/nova 1 kh1 2/product/Millipore
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nova 1 kh1 2 - by Bioz Stars, 2020-04
    85/100 stars
      Buy from Supplier

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    ( A ) Nitrocellulose filter binding assays of polycytidylate (C 17 ) RNA against wild-type hnRNP E1 KH1/2 protein and mutant protein harboring R(32)→K mutations in both KH domains. ( B ) against wild-type and Q(32)→R Nova-1 KH3 proteins].

    Journal: Nucleic Acids Research

    Article Title: Determination and augmentation of RNA sequence specificity of the Nova K-homology domains

    doi: 10.1093/nar/gkh799

    Figure Lengend Snippet: ( A ) Nitrocellulose filter binding assays of polycytidylate (C 17 ) RNA against wild-type hnRNP E1 KH1/2 protein and mutant protein harboring R(32)→K mutations in both KH domains. ( B ) against wild-type and Q(32)→R Nova-1 KH3 proteins].

    Article Snippet: An aliquot of 50 μl reactions containing 50–100 fmol of RNA body-labeled or end-labeled with 32 P and Nova-1 KH1/2, KH3 or hnRNP E1 proteins in 3-fold dilutions ranging from micromolar to nanomolar concentrations were mixed in buffer SBB and incubated for at least 10 min at room temperature, followed by filtering through 0.45 μm pore size nitrocellulose filters (Millipore) and washing.

    Techniques: Binding Assay, Mutagenesis

    Nitrocellulose filter binding assays of wild-type and mutant 10048 RNAs against Nova-1 KH1/2 protein. Each mutant 10048 RNA harbors a single nucleotide change in the UCAGUCAC sequence. The eight panels include all possible mutants in each of the eight positions of the UCAGUCAC sequence.

    Journal: Nucleic Acids Research

    Article Title: Determination and augmentation of RNA sequence specificity of the Nova K-homology domains

    doi: 10.1093/nar/gkh799

    Figure Lengend Snippet: Nitrocellulose filter binding assays of wild-type and mutant 10048 RNAs against Nova-1 KH1/2 protein. Each mutant 10048 RNA harbors a single nucleotide change in the UCAGUCAC sequence. The eight panels include all possible mutants in each of the eight positions of the UCAGUCAC sequence.

    Article Snippet: An aliquot of 50 μl reactions containing 50–100 fmol of RNA body-labeled or end-labeled with 32 P and Nova-1 KH1/2, KH3 or hnRNP E1 proteins in 3-fold dilutions ranging from micromolar to nanomolar concentrations were mixed in buffer SBB and incubated for at least 10 min at room temperature, followed by filtering through 0.45 μm pore size nitrocellulose filters (Millipore) and washing.

    Techniques: Binding Assay, Mutagenesis, Sequencing