Structured Review

TaKaRa mgcl2
Fluorescence intensity increment versus time of molecular beacon arrays immobilized on ( A ) a glutaraldehyde-derived glass slide and ( B ) an agarose film coated on a glass slide. The hybridization solution was 10 nM target T2 in 20 mM Tris–HCl pH 8.0 and 500 mM <t>MgCl2.</t>
Mgcl2, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 385 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mgcl2/product/TaKaRa
Average 94 stars, based on 385 article reviews
Price from $9.99 to $1999.99
mgcl2 - by Bioz Stars, 2020-08
94/100 stars

Images

1) Product Images from "Label-free hybridization detection of a single nucleotide mismatch by immobilization of molecular beacons on an agarose film"

Article Title: Label-free hybridization detection of a single nucleotide mismatch by immobilization of molecular beacons on an agarose film

Journal: Nucleic Acids Research

doi:

Fluorescence intensity increment versus time of molecular beacon arrays immobilized on ( A ) a glutaraldehyde-derived glass slide and ( B ) an agarose film coated on a glass slide. The hybridization solution was 10 nM target T2 in 20 mM Tris–HCl pH 8.0 and 500 mM MgCl2.
Figure Legend Snippet: Fluorescence intensity increment versus time of molecular beacon arrays immobilized on ( A ) a glutaraldehyde-derived glass slide and ( B ) an agarose film coated on a glass slide. The hybridization solution was 10 nM target T2 in 20 mM Tris–HCl pH 8.0 and 500 mM MgCl2.

Techniques Used: Fluorescence, Derivative Assay, Hybridization

Hybridization dynamics images of molecular beacon arrays immobilized on ( A ) a glutaraldehyde-derived glass slide and ( B ) an agarose film. The hybridization solution was 10 nM target T2 in 20 mM Tris–HCl pH 8.0 and 500 mM MgCl2.
Figure Legend Snippet: Hybridization dynamics images of molecular beacon arrays immobilized on ( A ) a glutaraldehyde-derived glass slide and ( B ) an agarose film. The hybridization solution was 10 nM target T2 in 20 mM Tris–HCl pH 8.0 and 500 mM MgCl2.

Techniques Used: Hybridization, Derivative Assay

Mismatch discrimination ratios of the different molecular beacons in bulk solution, immobilized on a glutaraldehyde-derived glass slide and on an agarose film, respectively. Discrimination ratio is defined as (PM – MM)/PM. PM is the fluorescence increment of perfectly matched probes and MM is the average fluorescence increment of three 1 base mismatched probes. The hybridization solution was 10 nM target T2 in 20 mM Tris–HCl pH 8.0, with different concentrations of MgCl2.
Figure Legend Snippet: Mismatch discrimination ratios of the different molecular beacons in bulk solution, immobilized on a glutaraldehyde-derived glass slide and on an agarose film, respectively. Discrimination ratio is defined as (PM – MM)/PM. PM is the fluorescence increment of perfectly matched probes and MM is the average fluorescence increment of three 1 base mismatched probes. The hybridization solution was 10 nM target T2 in 20 mM Tris–HCl pH 8.0, with different concentrations of MgCl2.

Techniques Used: Derivative Assay, Fluorescence, Hybridization

Related Articles

Polymerase Chain Reaction:

Article Title: Establishment of a Gene Detection System for Hotspot Mutations of Hearing Loss
Article Snippet: .. PCR and Melting Curve Analysis The reactions were performed in a 20 μ l solution containing HpH buffer [ ], 3.5 mM MgCl2 , 0.25 mM dNTPs, 1 U HS Taq DNA polymerase (TaKaRa, Dalian, China), 0.1 μ M probe, 0.05 μ M (Tube-A) or 0.2 μ M (Tube-B) of each specific primer, 0.125 μ M (Tube-A) or 0.2 μ M (Tube-B) forward universal primer, 1.25 μ M (Tube-A) or 2 μ M (Tube-B) reverse universal primer, 2 μ L aqueous template (extracted genomic DNA or pyrolysis saliva), or a 1.0 mm Harris manual micropunch (Sangerbio, Shanghai) to punch holes of FTA sample collection cards for sampling dried blood spots; a successful punch was obtained when the FTA disc was transferred from card to target well. ..

Article Title: Expression of Interleukin-1?, Tumor Necrosis Factor Alpha, and Interleukin-6 in Human Peripheral Blood Leukocytes Exposed to Human Granulocytic Ehrlichiosis Agent or Recombinant Major Surface Protein P44
Article Snippet: .. The cDNA (2 μl) was amplified in a 50-μl reaction mixture containing 1× PCR buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl2 ), 0.2 mM deoxynucleoside triphosphates, and 0.4 μM (each) 3′ and 5′ primers (Clontech Laboratories, Inc., Palo Alto, Calif.) in a DNA thermal cycler (model 480; Perkin-Elmer Corp., Norwalk, Conn.). .. Positive controls for all cytokines were obtained from Clontech.

Article Title: The effect of sodium butyrate and cisplatin on expression of EMT markers
Article Snippet: .. First PCR was carried out in a 20 μL mixture containing 10x Gold buffer without MgCl2 (2 μL), 25mM MgCl2 (2 μL), 2.5 mM dNTPs solution Takara (1.6 μL), 10 pmol/μl primers (1 μL), 5U/μL AmpliTaq Gold DNA Polymerase (0.3 μL) (Applied Biosystems, CA), water and 2 μL of bisulfite-converted DNA in the Veriti Thermocycler (Applied Biosystems, CA). ..

Article Title: Silencing Chitinase Genes Increases Susceptibility of Tetranychus cinnabarinus (Boisduval) to Scopoletin
Article Snippet: .. PCRs were performed with a 25 μ L reaction volume with 2.5 μ L 10x PCR buffer (Mg2+ -free), 2.0 μ L dNTPs (2.5 mM), 2.5 μ L MgCl2 (25 mM), 1 μ L cDNA templates, 1 μ L each primer (10 mM), 0.2 μ L rTaq™ polymerase (TaKaRa), and 14.8 μ L ddH2 O. PCR program was 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 48°C to 60°C (based on primer annealing temperatures) for 30 s, 72°C extension for 1 min to 2 min (based on the predicted length of amplified products), and a final extension of 10 min at 72°C. .. Amplified PCR fragments were gel-purified by using a gel extraction mini kit (Tiangen, Beijing, China), ligated into pMD™ 19-T vector (Takara, Dalian, China) and then transformed into Trans5α competent cells of Escherichia coli (Tiangen, Beijing, China).

Article Title: Genomic, RNA, and ecological divergences of the Revolver transposon-like multi-gene family in Triticeae
Article Snippet: .. Reaction mixtures contained 10 ng of template cDNA, 50 pmoles of each primer (5'-GGCACGAGGGTACGAGTCCGAG-3', 5'-GGCACAACTCATGTAAAAGAGGG-3'), 0.4 mM dNTPs, 1 × LA PCR buffer II, 2.5 mM MgCl2 , and 0.5 U of LA Taq polymerase (Takara) in a volume of 25 μL. .. The PCR reaction program consisted of 30 cycles of 30 sec at 95°C, 30 sec at 63°C, and 1 min at 72°C.

Amplification:

Article Title: Expression of Interleukin-1?, Tumor Necrosis Factor Alpha, and Interleukin-6 in Human Peripheral Blood Leukocytes Exposed to Human Granulocytic Ehrlichiosis Agent or Recombinant Major Surface Protein P44
Article Snippet: .. The cDNA (2 μl) was amplified in a 50-μl reaction mixture containing 1× PCR buffer (10 mM Tris-HCl [pH 8.3], 50 mM KCl, 1.5 mM MgCl2 ), 0.2 mM deoxynucleoside triphosphates, and 0.4 μM (each) 3′ and 5′ primers (Clontech Laboratories, Inc., Palo Alto, Calif.) in a DNA thermal cycler (model 480; Perkin-Elmer Corp., Norwalk, Conn.). .. Positive controls for all cytokines were obtained from Clontech.

Article Title: Silencing Chitinase Genes Increases Susceptibility of Tetranychus cinnabarinus (Boisduval) to Scopoletin
Article Snippet: .. PCRs were performed with a 25 μ L reaction volume with 2.5 μ L 10x PCR buffer (Mg2+ -free), 2.0 μ L dNTPs (2.5 mM), 2.5 μ L MgCl2 (25 mM), 1 μ L cDNA templates, 1 μ L each primer (10 mM), 0.2 μ L rTaq™ polymerase (TaKaRa), and 14.8 μ L ddH2 O. PCR program was 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 48°C to 60°C (based on primer annealing temperatures) for 30 s, 72°C extension for 1 min to 2 min (based on the predicted length of amplified products), and a final extension of 10 min at 72°C. .. Amplified PCR fragments were gel-purified by using a gel extraction mini kit (Tiangen, Beijing, China), ligated into pMD™ 19-T vector (Takara, Dalian, China) and then transformed into Trans5α competent cells of Escherichia coli (Tiangen, Beijing, China).

Sampling:

Article Title: Establishment of a Gene Detection System for Hotspot Mutations of Hearing Loss
Article Snippet: .. PCR and Melting Curve Analysis The reactions were performed in a 20 μ l solution containing HpH buffer [ ], 3.5 mM MgCl2 , 0.25 mM dNTPs, 1 U HS Taq DNA polymerase (TaKaRa, Dalian, China), 0.1 μ M probe, 0.05 μ M (Tube-A) or 0.2 μ M (Tube-B) of each specific primer, 0.125 μ M (Tube-A) or 0.2 μ M (Tube-B) forward universal primer, 1.25 μ M (Tube-A) or 2 μ M (Tube-B) reverse universal primer, 2 μ L aqueous template (extracted genomic DNA or pyrolysis saliva), or a 1.0 mm Harris manual micropunch (Sangerbio, Shanghai) to punch holes of FTA sample collection cards for sampling dried blood spots; a successful punch was obtained when the FTA disc was transferred from card to target well. ..

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 85
    TaKaRa atp adp reporter protein perceval
    Measurements of intracellular <t>ATP</t> and antioxidant capacity. ( A ) Confocal microscopy recordings of ATP in single cells. GD25β1 cells were transfected with <t>Perceval</t> and prepared as described in Material and Methods. The traces are representative recordings of two cells. The addition of 1 µM rotenone and the subsequent treatment with a combination of rotenone, sodium azide and the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) to maximally reduce ATP levels are indicated by the arrows. Quantifications from 8 cells are shown to the right. The bars show means of the lowest ATP level during treatment with rotenone alone and the ATP level just before addition of the inhibitor combination. ( B ) ATP levels of serum-starved BJ hTERT and GD25β1 cells after 45 min treatment with rotenone as determined by a colorimetric ATP assay performed described in Materials and Methods . n = 2. ( C ) Total antioxidant capacity (TAC) of serum-starved BJ hTERT and GD25β1 cells as determined by a colorimetric TAC assay described in Materials and Methods . The results are expressed as Trolox equivalents and were normalized to BJ hTERT cells in order to be able to compare the two cell lines with each other. n = 3. All error bars in Fig. 5 represent standard deviation, * p
    Atp Adp Reporter Protein Perceval, supplied by TaKaRa, 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/atp adp reporter protein perceval/product/TaKaRa
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    atp adp reporter protein perceval - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    85
    TaKaRa standard recombinase buffer
    Schematic of a typical recombination reaction in the Creator Splice system. The SD/intron, shown in purple, is the only difference between a Creator Splice acceptor and a standard acceptor vector. A Creator Splice donor and acceptor are recombined in the presence of Cre <t>recombinase</t> in vitro . An intron is formed in the resulting expression vector starting from the SD/intron supplied by the acceptor and ending with the splice acceptor from the donor vector. Upon transfection and transcription in mammalian cells, the intron is removed and the tag is juxtaposed onto the ORF. Abbreviations: bac – bacteria promoter, CmR – Chloramphenicol resistance ORF, kan/neoR – kanamycin and neomycin resistance gene, P – promoter, SA – splice acceptor, SD – splice donor.
    Standard Recombinase Buffer, supplied by TaKaRa, 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/standard recombinase buffer/product/TaKaRa
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    standard recombinase buffer - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    90
    TaKaRa mdn1 atp site mutants
    Rbins Inhibit <t>Mdn1’s</t> ATPase Activity In Vitro (A) Schematic for strategy used to purify recombinant full-length Mdn1. (B) The size-exclusion chromatography profile for Mdn1 (black trace). The activity of different fractions from this chromatography step was analyzed using a radioactive ATPase assay <t>([MgATP]</t> = 0.1 mM). Activity was normalized to the most active fraction (red dots, each fraction; trace, interpolation). (C) SDS-PAGE analysis of purified full-length Mdn1 (WT) (Coomassie stain). (D) Peptides identified by mass spectrometry of the purified protein are indicated (green bars, schematic generated using Proteome Discoverer 1.4, Thermo Scientific). (E) Mdn1’s activity in the presence of Rbin analogs (1 μM) was tested using an NADH-coupled ATPase assay ([MgATP] = 1 mM). The relative activity is indicated (mean ± range, n = 2 independent experiments). (F) Dose-dependent inhibition of the steady-state ATPase activity of wild-type Mdn1 and Mdn1(F1093L) by Rbin-2 (mean ± SD, n = 4 independent experiments). An apparent EC 50 was estimated for the inhibition of wild-type Mdn1 using a sigmoidal dose-response curve. Using similar equations, we were unable to properly fit the small decrease in activity of Mdn1(F1093L) across the concentration range tested. Unpaired t test (without Welch’s correction) of the measured activity for WT and Mdn1(F1093), at the highest three inhibitor concentrations tested, indicate that the difference in values are statistically significant (p = 0.0020 (at 16.7 μM), p = 0.0021 (at 3.33 μM), and p = 0.0019 (at 1.0 μM)). (G) The ATPase activity of Mdn1 in the presence Rbin-1 (1 μM) or AMP-PNP (2 mM) using the radioactive ATPase assay (MgATP = 0.1 mM). Error bars show SD (n = 6 independent experiments). (H) SDS-PAGE analysis of purified full-length Mdn1(F1093L) (Coomassie stain). See also Figure S4 .
    Mdn1 Atp Site Mutants, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mdn1 atp site mutants/product/TaKaRa
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mdn1 atp site mutants - by Bioz Stars, 2020-08
    90/100 stars
      Buy from Supplier

    95
    TaKaRa pcr buffer
    Epstein–Barr virus (EBV) typing of normal individuals and patients with systemic lupus erythematosus (SLE) in mouthwash samples. (a) <t>PCR/Southern</t> blot of the EBV nuclear antigen (EBNA)-3C encoding region for the cell lines carrying type 1 (ES-1, B95-8, LCL2, and Namalwa) and type 2 (SNU-99 and AG876) EBV. <t>DNA</t> extracted from each EBV infected cell line (5 ng) was subjected to EBNA-3C-specific PCR/Southern blot. PCR amplified products were transferred to a membrane and hybridized with an EBNA-3C probe common to both type 1 and type 2 EBV. The expected PCR product sizes were 153 bp for type 1 EBV and 246 bp for type 2 EBV. The EBV negative cell line BJAB and distilled water served as negative controls. (b,c) PCR/Southern blot of the EBNA-3C encoding region for the DNA from mouthwash samples. One 20th of the DNA isolated from mouthwash samples was used for each PCR reaction. Representative results obtained from normal controls (panel b) and SLE patients (panel c) are shown. Namalwa and AG876 were used as positive controls for type 1 and type 2 EBV, respectively. Distilled water (dH 2 0) and DNA isolated from BJAB were used as negative controls.
    Pcr Buffer, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 1840 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr buffer/product/TaKaRa
    Average 95 stars, based on 1840 article reviews
    Price from $9.99 to $1999.99
    pcr buffer - by Bioz Stars, 2020-08
    95/100 stars
      Buy from Supplier

    Image Search Results


    Measurements of intracellular ATP and antioxidant capacity. ( A ) Confocal microscopy recordings of ATP in single cells. GD25β1 cells were transfected with Perceval and prepared as described in Material and Methods. The traces are representative recordings of two cells. The addition of 1 µM rotenone and the subsequent treatment with a combination of rotenone, sodium azide and the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) to maximally reduce ATP levels are indicated by the arrows. Quantifications from 8 cells are shown to the right. The bars show means of the lowest ATP level during treatment with rotenone alone and the ATP level just before addition of the inhibitor combination. ( B ) ATP levels of serum-starved BJ hTERT and GD25β1 cells after 45 min treatment with rotenone as determined by a colorimetric ATP assay performed described in Materials and Methods . n = 2. ( C ) Total antioxidant capacity (TAC) of serum-starved BJ hTERT and GD25β1 cells as determined by a colorimetric TAC assay described in Materials and Methods . The results are expressed as Trolox equivalents and were normalized to BJ hTERT cells in order to be able to compare the two cell lines with each other. n = 3. All error bars in Fig. 5 represent standard deviation, * p

    Journal: PLoS ONE

    Article Title: The Role of Mechanical Force and ROS in Integrin-Dependent Signals

    doi: 10.1371/journal.pone.0064897

    Figure Lengend Snippet: Measurements of intracellular ATP and antioxidant capacity. ( A ) Confocal microscopy recordings of ATP in single cells. GD25β1 cells were transfected with Perceval and prepared as described in Material and Methods. The traces are representative recordings of two cells. The addition of 1 µM rotenone and the subsequent treatment with a combination of rotenone, sodium azide and the glycolysis inhibitor 2-deoxy-D-glucose (2-DG) to maximally reduce ATP levels are indicated by the arrows. Quantifications from 8 cells are shown to the right. The bars show means of the lowest ATP level during treatment with rotenone alone and the ATP level just before addition of the inhibitor combination. ( B ) ATP levels of serum-starved BJ hTERT and GD25β1 cells after 45 min treatment with rotenone as determined by a colorimetric ATP assay performed described in Materials and Methods . n = 2. ( C ) Total antioxidant capacity (TAC) of serum-starved BJ hTERT and GD25β1 cells as determined by a colorimetric TAC assay described in Materials and Methods . The results are expressed as Trolox equivalents and were normalized to BJ hTERT cells in order to be able to compare the two cell lines with each other. n = 3. All error bars in Fig. 5 represent standard deviation, * p

    Article Snippet: Measurements of intracellular ATP a) Single cell analysis: GD25β1 cells were transfected to transiently express the fluorescent ATP/ADP reporter protein Perceval , and cytosolic mCherry as an internal standard (pmCherry-C1, #632524, Clontech).

    Techniques: Confocal Microscopy, Transfection, ATP Assay, Standard Deviation

    Schematic of a typical recombination reaction in the Creator Splice system. The SD/intron, shown in purple, is the only difference between a Creator Splice acceptor and a standard acceptor vector. A Creator Splice donor and acceptor are recombined in the presence of Cre recombinase in vitro . An intron is formed in the resulting expression vector starting from the SD/intron supplied by the acceptor and ending with the splice acceptor from the donor vector. Upon transfection and transcription in mammalian cells, the intron is removed and the tag is juxtaposed onto the ORF. Abbreviations: bac – bacteria promoter, CmR – Chloramphenicol resistance ORF, kan/neoR – kanamycin and neomycin resistance gene, P – promoter, SA – splice acceptor, SD – splice donor.

    Journal: BMC Biotechnology

    Article Title: Modification of the Creator recombination system for proteomics applications - improved expression by addition of splice sites

    doi: 10.1186/1472-6750-6-13

    Figure Lengend Snippet: Schematic of a typical recombination reaction in the Creator Splice system. The SD/intron, shown in purple, is the only difference between a Creator Splice acceptor and a standard acceptor vector. A Creator Splice donor and acceptor are recombined in the presence of Cre recombinase in vitro . An intron is formed in the resulting expression vector starting from the SD/intron supplied by the acceptor and ending with the splice acceptor from the donor vector. Upon transfection and transcription in mammalian cells, the intron is removed and the tag is juxtaposed onto the ORF. Abbreviations: bac – bacteria promoter, CmR – Chloramphenicol resistance ORF, kan/neoR – kanamycin and neomycin resistance gene, P – promoter, SA – splice acceptor, SD – splice donor.

    Article Snippet: For recombination reactions, 400 ng of donor and acceptor were recombined in a final volume of 20 μl for 15 minutes at room temperature in the presence of Cre recombinase and either optimized recombinase buffer (5 mM TrisHCl pH7.9, 3.3 mM NaCl, 1 mM MgCl2 and 1 mM spermidine) or standard recombinase buffer (50 mM TrisHCl pH7.9, 33 mM NaCl, 10 mM MgCl2 ) according to Clontech manual PT3460-1.

    Techniques: Plasmid Preparation, In Vitro, Expressing, Transfection, BAC Assay

    Rbins Inhibit Mdn1’s ATPase Activity In Vitro (A) Schematic for strategy used to purify recombinant full-length Mdn1. (B) The size-exclusion chromatography profile for Mdn1 (black trace). The activity of different fractions from this chromatography step was analyzed using a radioactive ATPase assay ([MgATP] = 0.1 mM). Activity was normalized to the most active fraction (red dots, each fraction; trace, interpolation). (C) SDS-PAGE analysis of purified full-length Mdn1 (WT) (Coomassie stain). (D) Peptides identified by mass spectrometry of the purified protein are indicated (green bars, schematic generated using Proteome Discoverer 1.4, Thermo Scientific). (E) Mdn1’s activity in the presence of Rbin analogs (1 μM) was tested using an NADH-coupled ATPase assay ([MgATP] = 1 mM). The relative activity is indicated (mean ± range, n = 2 independent experiments). (F) Dose-dependent inhibition of the steady-state ATPase activity of wild-type Mdn1 and Mdn1(F1093L) by Rbin-2 (mean ± SD, n = 4 independent experiments). An apparent EC 50 was estimated for the inhibition of wild-type Mdn1 using a sigmoidal dose-response curve. Using similar equations, we were unable to properly fit the small decrease in activity of Mdn1(F1093L) across the concentration range tested. Unpaired t test (without Welch’s correction) of the measured activity for WT and Mdn1(F1093), at the highest three inhibitor concentrations tested, indicate that the difference in values are statistically significant (p = 0.0020 (at 16.7 μM), p = 0.0021 (at 3.33 μM), and p = 0.0019 (at 1.0 μM)). (G) The ATPase activity of Mdn1 in the presence Rbin-1 (1 μM) or AMP-PNP (2 mM) using the radioactive ATPase assay (MgATP = 0.1 mM). Error bars show SD (n = 6 independent experiments). (H) SDS-PAGE analysis of purified full-length Mdn1(F1093L) (Coomassie stain). See also Figure S4 .

    Journal: Cell

    Article Title: Potent, Reversible, and Specific Chemical Inhibitors of Eukaryotic Ribosome Biogenesis

    doi: 10.1016/j.cell.2016.08.070

    Figure Lengend Snippet: Rbins Inhibit Mdn1’s ATPase Activity In Vitro (A) Schematic for strategy used to purify recombinant full-length Mdn1. (B) The size-exclusion chromatography profile for Mdn1 (black trace). The activity of different fractions from this chromatography step was analyzed using a radioactive ATPase assay ([MgATP] = 0.1 mM). Activity was normalized to the most active fraction (red dots, each fraction; trace, interpolation). (C) SDS-PAGE analysis of purified full-length Mdn1 (WT) (Coomassie stain). (D) Peptides identified by mass spectrometry of the purified protein are indicated (green bars, schematic generated using Proteome Discoverer 1.4, Thermo Scientific). (E) Mdn1’s activity in the presence of Rbin analogs (1 μM) was tested using an NADH-coupled ATPase assay ([MgATP] = 1 mM). The relative activity is indicated (mean ± range, n = 2 independent experiments). (F) Dose-dependent inhibition of the steady-state ATPase activity of wild-type Mdn1 and Mdn1(F1093L) by Rbin-2 (mean ± SD, n = 4 independent experiments). An apparent EC 50 was estimated for the inhibition of wild-type Mdn1 using a sigmoidal dose-response curve. Using similar equations, we were unable to properly fit the small decrease in activity of Mdn1(F1093L) across the concentration range tested. Unpaired t test (without Welch’s correction) of the measured activity for WT and Mdn1(F1093), at the highest three inhibitor concentrations tested, indicate that the difference in values are statistically significant (p = 0.0020 (at 16.7 μM), p = 0.0021 (at 3.33 μM), and p = 0.0019 (at 1.0 μM)). (G) The ATPase activity of Mdn1 in the presence Rbin-1 (1 μM) or AMP-PNP (2 mM) using the radioactive ATPase assay (MgATP = 0.1 mM). Error bars show SD (n = 6 independent experiments). (H) SDS-PAGE analysis of purified full-length Mdn1(F1093L) (Coomassie stain). See also Figure S4 .

    Article Snippet: For the mdn1 ATP site mutants (e. g. Walker A, Walker B, and R finger), mutations were introduced into N (1-3355 bp) or M (3350-8528 bp) fragment of the mdn1 gene using PrimeSTAR Mutagenesis Basal Kit (TAKARA), followed by ligation of N, M, and C (8523-14154 bp) fragments.

    Techniques: Activity Assay, In Vitro, Recombinant, Size-exclusion Chromatography, Chromatography, ATPase Assay, SDS Page, Purification, Staining, Mass Spectrometry, Generated, Inhibition, Concentration Assay

    Epstein–Barr virus (EBV) typing of normal individuals and patients with systemic lupus erythematosus (SLE) in mouthwash samples. (a) PCR/Southern blot of the EBV nuclear antigen (EBNA)-3C encoding region for the cell lines carrying type 1 (ES-1, B95-8, LCL2, and Namalwa) and type 2 (SNU-99 and AG876) EBV. DNA extracted from each EBV infected cell line (5 ng) was subjected to EBNA-3C-specific PCR/Southern blot. PCR amplified products were transferred to a membrane and hybridized with an EBNA-3C probe common to both type 1 and type 2 EBV. The expected PCR product sizes were 153 bp for type 1 EBV and 246 bp for type 2 EBV. The EBV negative cell line BJAB and distilled water served as negative controls. (b,c) PCR/Southern blot of the EBNA-3C encoding region for the DNA from mouthwash samples. One 20th of the DNA isolated from mouthwash samples was used for each PCR reaction. Representative results obtained from normal controls (panel b) and SLE patients (panel c) are shown. Namalwa and AG876 were used as positive controls for type 1 and type 2 EBV, respectively. Distilled water (dH 2 0) and DNA isolated from BJAB were used as negative controls.

    Journal: Arthritis Research & Therapy

    Article Title: Patients with systemic lupus erythematosus have abnormally elevated Epstein-Barr virus load in blood

    doi: 10.1186/ar1181

    Figure Lengend Snippet: Epstein–Barr virus (EBV) typing of normal individuals and patients with systemic lupus erythematosus (SLE) in mouthwash samples. (a) PCR/Southern blot of the EBV nuclear antigen (EBNA)-3C encoding region for the cell lines carrying type 1 (ES-1, B95-8, LCL2, and Namalwa) and type 2 (SNU-99 and AG876) EBV. DNA extracted from each EBV infected cell line (5 ng) was subjected to EBNA-3C-specific PCR/Southern blot. PCR amplified products were transferred to a membrane and hybridized with an EBNA-3C probe common to both type 1 and type 2 EBV. The expected PCR product sizes were 153 bp for type 1 EBV and 246 bp for type 2 EBV. The EBV negative cell line BJAB and distilled water served as negative controls. (b,c) PCR/Southern blot of the EBNA-3C encoding region for the DNA from mouthwash samples. One 20th of the DNA isolated from mouthwash samples was used for each PCR reaction. Representative results obtained from normal controls (panel b) and SLE patients (panel c) are shown. Namalwa and AG876 were used as positive controls for type 1 and type 2 EBV, respectively. Distilled water (dH 2 0) and DNA isolated from BJAB were used as negative controls.

    Article Snippet: PCR was performed in a total volume of 10 μl, which contained 2 μl extracted DNA sample, 1 μl 10× PCR buffer (with 100 mmol/l Tris-HCl, 500 mmol/l KCl, and 15 mmol/l MgCl2 ), 2 μl primer pair mix, and 1 U Taq polymerase (Takara, Tokyo, Japan).

    Techniques: Polymerase Chain Reaction, Southern Blot, Infection, Amplification, Isolation

    Epstein–Barr virus (EBV) loads in peripheral blood mononuclear cells (PBMCs) from 29 normal individuals and 24 patients with systemic lupus erythematosus (SLE). (a) Sensitivity of PCR/Southern blot for the EBV nuclear antigen (EBNA)-3C sequence. DNA was purified from serial 10-fold dilutions of Namalwa cells (corresponding to 1 to 1 × 10 7 cells) were mixed with BJAB cells to yield a total cell number of 1 × 10 7 . PCR was performed using a 100th of the purified DNA (corresponding to DNA of 10 5 cells). The PCR products were separated in an agarose gel, transferred to a membrane, and probed with an EBNA-3C-specific oligonucleotide. (b) EBV loads of normal individuals and SLE patients. The mean EBV load of each group is presented as a heavy horizontal line.

    Journal: Arthritis Research & Therapy

    Article Title: Patients with systemic lupus erythematosus have abnormally elevated Epstein-Barr virus load in blood

    doi: 10.1186/ar1181

    Figure Lengend Snippet: Epstein–Barr virus (EBV) loads in peripheral blood mononuclear cells (PBMCs) from 29 normal individuals and 24 patients with systemic lupus erythematosus (SLE). (a) Sensitivity of PCR/Southern blot for the EBV nuclear antigen (EBNA)-3C sequence. DNA was purified from serial 10-fold dilutions of Namalwa cells (corresponding to 1 to 1 × 10 7 cells) were mixed with BJAB cells to yield a total cell number of 1 × 10 7 . PCR was performed using a 100th of the purified DNA (corresponding to DNA of 10 5 cells). The PCR products were separated in an agarose gel, transferred to a membrane, and probed with an EBNA-3C-specific oligonucleotide. (b) EBV loads of normal individuals and SLE patients. The mean EBV load of each group is presented as a heavy horizontal line.

    Article Snippet: PCR was performed in a total volume of 10 μl, which contained 2 μl extracted DNA sample, 1 μl 10× PCR buffer (with 100 mmol/l Tris-HCl, 500 mmol/l KCl, and 15 mmol/l MgCl2 ), 2 μl primer pair mix, and 1 U Taq polymerase (Takara, Tokyo, Japan).

    Techniques: Polymerase Chain Reaction, Southern Blot, Sequencing, Purification, Agarose Gel Electrophoresis