s2 cell transfections  (Qiagen)

 
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    QIAGEN Plasmid Midi Kit
    Description:
    For purification of up to 10 mg transfection grade plasmid or cosmid DNA Kit contents Qiagen Plasmid Midi Kit 25 preps 25 to 100mL Culture Volume Manual Centrifugation Processing 150 min Time Run Anion exchange Technology 1 Sample Run For Purification of up to 100g Transfection grade Plasmid or Cosmid DNA Ideal for Cloning Sequencing Capillary Sequencing In vitro Transcription Includes 25 Qiagen to tip 100 Reagents Buffers Benefits Purity equivalent to 2 x CsCl gradient centrifugation High yields of plasmid DNA Cost effective preparations LyseBlue for optimum lysis and maximum DNA yie
    Catalog Number:
    12143
    Price:
    288
    Category:
    QIAGEN Plasmid Kits
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    Structured Review

    Qiagen s2 cell transfections
    QIAGEN Plasmid Midi Kit
    For purification of up to 10 mg transfection grade plasmid or cosmid DNA Kit contents Qiagen Plasmid Midi Kit 25 preps 25 to 100mL Culture Volume Manual Centrifugation Processing 150 min Time Run Anion exchange Technology 1 Sample Run For Purification of up to 100g Transfection grade Plasmid or Cosmid DNA Ideal for Cloning Sequencing Capillary Sequencing In vitro Transcription Includes 25 Qiagen to tip 100 Reagents Buffers Benefits Purity equivalent to 2 x CsCl gradient centrifugation High yields of plasmid DNA Cost effective preparations LyseBlue for optimum lysis and maximum DNA yie
    https://www.bioz.com/result/s2 cell transfections/product/Qiagen
    Average 85 stars, based on 11306 article reviews
    Price from $9.99 to $1999.99
    s2 cell transfections - by Bioz Stars, 2020-08
    85/100 stars

    Images

    1) Product Images from "BAcTrace a new tool for retrograde tracing of neuronal circuits"

    Article Title: BAcTrace a new tool for retrograde tracing of neuronal circuits

    Journal: bioRxiv

    doi: 10.1101/2020.01.24.918656

    Testing BAcTrace in tissue culture. ( A ) Western blot analysis of S2 cell extracts after transfection and incubation with decreasing amounts of toxin. At around 0.3pM half of the hSNAP25 appears to be cleaved. The empty arrowhead indicates un-cleaved products while the solid arrowhead indicates cleaved products. ( B ) Flag::hSNAP25 cleavage efficiency does not change at the tested concentrations of hTfR::GFP receptor. ( C ) Schematics of TEV constructs used in (D) and (G). ( D ) Immunochemistry of S2 cells transfected with the indicated sensors and TEV variants. Solid arrowheads indicate non-cleaved sensor on the plasma membrane and empty arrowheads indicate cleaved sensor on the plasma membrane as determined by the presence of red and absence of green fluorescence. ( E ) Prediction of N-glycosilation sites on TEV [ 21 ]. ( F ) TEV structure (1lvb, [ 43 ]) with potential glycosilation sites from F highlighted in blue. Bound pseudosubstrate is shown in yellow. ( G ) Western blot against CD2 showing that only the TEV T173V is capable of cleaving the TEV sensor. The band marked with an asterix is non-specific. Band marked with a solid black arrowhead corresponds to the TEV sensor. Epitopes detected by antibodies are indicated in bold in (A), (B), (D) and (G).
    Figure Legend Snippet: Testing BAcTrace in tissue culture. ( A ) Western blot analysis of S2 cell extracts after transfection and incubation with decreasing amounts of toxin. At around 0.3pM half of the hSNAP25 appears to be cleaved. The empty arrowhead indicates un-cleaved products while the solid arrowhead indicates cleaved products. ( B ) Flag::hSNAP25 cleavage efficiency does not change at the tested concentrations of hTfR::GFP receptor. ( C ) Schematics of TEV constructs used in (D) and (G). ( D ) Immunochemistry of S2 cells transfected with the indicated sensors and TEV variants. Solid arrowheads indicate non-cleaved sensor on the plasma membrane and empty arrowheads indicate cleaved sensor on the plasma membrane as determined by the presence of red and absence of green fluorescence. ( E ) Prediction of N-glycosilation sites on TEV [ 21 ]. ( F ) TEV structure (1lvb, [ 43 ]) with potential glycosilation sites from F highlighted in blue. Bound pseudosubstrate is shown in yellow. ( G ) Western blot against CD2 showing that only the TEV T173V is capable of cleaving the TEV sensor. The band marked with an asterix is non-specific. Band marked with a solid black arrowhead corresponds to the TEV sensor. Epitopes detected by antibodies are indicated in bold in (A), (B), (D) and (G).

    Techniques Used: Western Blot, Transfection, Incubation, Construct, Fluorescence

    Testing BAcTrace in tissue culture. ( A ) Schematic of transgenes and tissue culture experimental outline used to test E. coli produced toxin. ( B ) Western blot analysis of S2 cell extracts after transfection and incubation with toxin made in bacteria. Empty arrowheads indicate un-cleaved and solid arrowheads cleaved toxin sensors. Toxin concentrations (nM) in each lane are: 0, 1.7, 3.4, 6.9, 14, 0, 1.7, 3.4, 6.9 and 14. Smaller band in Tomato::HA panels is a degradation C-terminal fragment. ( C ) Schematic of cell mixing experiments to test toxin produced in insect cells. ( D ) Western blot analysis of S2 cell extracts from cell mixing experiments. Conditions marked with * correspond to those from panel (C). Epitopes detected by antibodies are indicated in bold in (B) and (D).
    Figure Legend Snippet: Testing BAcTrace in tissue culture. ( A ) Schematic of transgenes and tissue culture experimental outline used to test E. coli produced toxin. ( B ) Western blot analysis of S2 cell extracts after transfection and incubation with toxin made in bacteria. Empty arrowheads indicate un-cleaved and solid arrowheads cleaved toxin sensors. Toxin concentrations (nM) in each lane are: 0, 1.7, 3.4, 6.9, 14, 0, 1.7, 3.4, 6.9 and 14. Smaller band in Tomato::HA panels is a degradation C-terminal fragment. ( C ) Schematic of cell mixing experiments to test toxin produced in insect cells. ( D ) Western blot analysis of S2 cell extracts from cell mixing experiments. Conditions marked with * correspond to those from panel (C). Epitopes detected by antibodies are indicated in bold in (B) and (D).

    Techniques Used: Produced, Western Blot, Transfection, Incubation

    2) Product Images from "Integron Carrying a Novel Metallo-?-Lactamase Gene, blaIMP-16, and a Fused Form of Aminoglycoside-Resistant Gene aac(6′)-30/aac(6′)-Ib′: Report from the SENTRY Antimicrobial Surveillance Program"

    Article Title: Integron Carrying a Novel Metallo-?-Lactamase Gene, blaIMP-16, and a Fused Form of Aminoglycoside-Resistant Gene aac(6′)-30/aac(6′)-Ib′: Report from the SENTRY Antimicrobial Surveillance Program

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.48.12.4693-4702.2004

    Schematic representation of the class 1 integron-containing bla IMP-16 gene cassette from clinical isolate P. aeruginosa 101-4704. Boxes, inserted genes; arrows, transcriptional orientations; black circles, 59-be's; white circle, attI1 recombination site; lines, DNA of the inserts contained within recombinant plasmids pREM-1, pREM-2, pREM-3, and pREM-4; arrowheads, primer positions and their orientations; M, start codon; asterisk, the location of the stop codon for the particular gene.
    Figure Legend Snippet: Schematic representation of the class 1 integron-containing bla IMP-16 gene cassette from clinical isolate P. aeruginosa 101-4704. Boxes, inserted genes; arrows, transcriptional orientations; black circles, 59-be's; white circle, attI1 recombination site; lines, DNA of the inserts contained within recombinant plasmids pREM-1, pREM-2, pREM-3, and pREM-4; arrowheads, primer positions and their orientations; M, start codon; asterisk, the location of the stop codon for the particular gene.

    Techniques Used: Recombinant

    3) Product Images from "Effects of optional structural elements, including two alternative amino termini and a new splicing cassette IV, on the function of the sodium–bicarbonate cotransporter NBCn1 (SLC4A7)"

    Article Title: Effects of optional structural elements, including two alternative amino termini and a new splicing cassette IV, on the function of the sodium–bicarbonate cotransporter NBCn1 (SLC4A7)

    Journal: The Journal of Physiology

    doi: 10.1113/jphysiol.2013.258673

    Summary of pH i recovery rates (dpH i /d t ) of oocytes expressing human NBCn1 EGFP-tagged constructs ( A ), untagged constructs ( B ) or various NBCn1-G constructs ( C )
    Figure Legend Snippet: Summary of pH i recovery rates (dpH i /d t ) of oocytes expressing human NBCn1 EGFP-tagged constructs ( A ), untagged constructs ( B ) or various NBCn1-G constructs ( C )

    Techniques Used: Expressing, Construct

    Cloning and expression patterns of NBCn1 in human and mouse tissues
    Figure Legend Snippet: Cloning and expression patterns of NBCn1 in human and mouse tissues

    Techniques Used: Clone Assay, Expressing

    Western blots of total ( A ) and surface ( B ) NBCn1, and summary of relative surface abundance ( C ) of mouse NBCn1 variants in Xenopus oocytes
    Figure Legend Snippet: Western blots of total ( A ) and surface ( B ) NBCn1, and summary of relative surface abundance ( C ) of mouse NBCn1 variants in Xenopus oocytes

    Techniques Used: Western Blot

    Cloning and expression patterns of NBCn1 in human and mouse tissues
    Figure Legend Snippet: Cloning and expression patterns of NBCn1 in human and mouse tissues

    Techniques Used: Clone Assay, Expressing

    Summary of pH i recovery rates (dpH i /d t ) of oocytes expressing different mouse NBCn1 variants
    Figure Legend Snippet: Summary of pH i recovery rates (dpH i /d t ) of oocytes expressing different mouse NBCn1 variants

    Techniques Used: Expressing

    NBCn1-dependent functional expression ( A ) and intrinsic HCO 3 − transport activity ( B )
    Figure Legend Snippet: NBCn1-dependent functional expression ( A ) and intrinsic HCO 3 − transport activity ( B )

    Techniques Used: Functional Assay, Expressing, Activity Assay

    Representative recordings of intracellular pH (pH i ) and membrane potential ( V m ) from oocytes expressing human NBCn1-E-EGFP ( A ), NBCn1-G-EGFP ( B ) or H 2 O-injected control oocytes ( C )
    Figure Legend Snippet: Representative recordings of intracellular pH (pH i ) and membrane potential ( V m ) from oocytes expressing human NBCn1-E-EGFP ( A ), NBCn1-G-EGFP ( B ) or H 2 O-injected control oocytes ( C )

    Techniques Used: Expressing, Injection

    Cloning and expression patterns of NBCn1 in human and mouse tissues
    Figure Legend Snippet: Cloning and expression patterns of NBCn1 in human and mouse tissues

    Techniques Used: Clone Assay, Expressing

    Summary of known major NBCn1 variants
    Figure Legend Snippet: Summary of known major NBCn1 variants

    Techniques Used:

    PCR cloning of NBCn1 transcripts from human ( A and B ) and mouse ( C ) tissues
    Figure Legend Snippet: PCR cloning of NBCn1 transcripts from human ( A and B ) and mouse ( C ) tissues

    Techniques Used: Polymerase Chain Reaction, Clone Assay

    4) Product Images from "MID1 and MID2 homo- and heterodimerise to tether the rapamycin-sensitive PP2A regulatory subunit, Alpha 4, to microtubules: implications for the clinical variability of X-linked Opitz GBBB syndrome and other developmental disorders"

    Article Title: MID1 and MID2 homo- and heterodimerise to tether the rapamycin-sensitive PP2A regulatory subunit, Alpha 4, to microtubules: implications for the clinical variability of X-linked Opitz GBBB syndrome and other developmental disorders

    Journal: BMC Cell Biology

    doi: 10.1186/1471-2121-3-1

    Alpha 4 interacts with MID1 and MID2. (A) Yeast two-hybrid analysis of the interaction between MID1 and Alpha 4 as well as MID2 and Alpha 4. Yeast agar plate (leu - trp - his - , 75 mM 3-AT) showing growth for MID1/Alpha 4 and MID2/Alpha 4 interactions as well as positive control two-hybrid combinations and no growth for negative controls. (B) Detection of full-length myc tagged-Alpha 4 when co-expressed with GFP-MID1 and GFP-MID2 fusion proteins in transiently transfected Cos1 cells. (a) GFP-MID1 fluorescence (green), (b) anti-myc antibody detecting myc-Alpha 4 localisation (red), (c) overlay of (a), (b) showing co-localisation of the myc-Alpha 4 fusion protein and GFP-MID1, with DAPI stain for DNA (blue). (d) GFP-MID2 fluorescence (green), (e) myc-Alpha 4 localisation (using same detection as b) (red), (f) overlay of (d), (e) with DAPI (blue) showing co-localisation of the myc-Alpha 4 fusion protein and GFP-MID2. (g) Detection of transiently expressed myc-Alpha 4 fusion protein in Cos1 cells, (h) overlay of (g) and DAPI stain showing cytoplasmic distribution of myc-Alpha 4 fusion protein.
    Figure Legend Snippet: Alpha 4 interacts with MID1 and MID2. (A) Yeast two-hybrid analysis of the interaction between MID1 and Alpha 4 as well as MID2 and Alpha 4. Yeast agar plate (leu - trp - his - , 75 mM 3-AT) showing growth for MID1/Alpha 4 and MID2/Alpha 4 interactions as well as positive control two-hybrid combinations and no growth for negative controls. (B) Detection of full-length myc tagged-Alpha 4 when co-expressed with GFP-MID1 and GFP-MID2 fusion proteins in transiently transfected Cos1 cells. (a) GFP-MID1 fluorescence (green), (b) anti-myc antibody detecting myc-Alpha 4 localisation (red), (c) overlay of (a), (b) showing co-localisation of the myc-Alpha 4 fusion protein and GFP-MID1, with DAPI stain for DNA (blue). (d) GFP-MID2 fluorescence (green), (e) myc-Alpha 4 localisation (using same detection as b) (red), (f) overlay of (d), (e) with DAPI (blue) showing co-localisation of the myc-Alpha 4 fusion protein and GFP-MID2. (g) Detection of transiently expressed myc-Alpha 4 fusion protein in Cos1 cells, (h) overlay of (g) and DAPI stain showing cytoplasmic distribution of myc-Alpha 4 fusion protein.

    Techniques Used: Positive Control, Transfection, Fluorescence, Staining

    5) Product Images from "An enhanced toolkit for the generation of knockout and marker-free fluorescentPlasmodium chabaudi"

    Article Title: An enhanced toolkit for the generation of knockout and marker-free fluorescentPlasmodium chabaudi

    Journal: Wellcome Open Research

    doi: 10.12688/wellcomeopenres.15587.1

    Generation of Plasmo GEM resources for genetic modification of P. chabaudi. Insert-size distribution for P. chabaudi genomic DNA (gDNA) clone library showing number of clones for each genomic insert-size category, with an average genomic clone size of 6-7 kb ( A ). Size selected P. chabaudi genomic DNA fragments ( ii ) were cloned into the pJAZZ-OK Blunt vector ( i ) to generate the PcG01 and PcG02 genomic libraries. The pJAZZ-OK Blunt vector encode hairpin telomers (shown in black), a telomerase gene (TelN), replication factor and origin (repA), regulator of replication (cB) and a kanamycin resistance gene (kanR). Genomic library clones are converted into gene targeting vectors by recombineering, which introduces a dual bacterial selectable marker zeocin-pheS (zeo-pheS), ( iii ). Linear transfection-ready vectors are then generated by Gateway cloning, which facilitates the exchange of the zeo-pheS cassette for the parasite positive-negative selection marker human dihydrofolate reductase-uridyl phosphoribosyl transferase (hdhfr-yfcu), ( iv ) that are prepared for transfection by NotI digest and integrate with high efficiency into the P. chabaudi genome ( v ).
    Figure Legend Snippet: Generation of Plasmo GEM resources for genetic modification of P. chabaudi. Insert-size distribution for P. chabaudi genomic DNA (gDNA) clone library showing number of clones for each genomic insert-size category, with an average genomic clone size of 6-7 kb ( A ). Size selected P. chabaudi genomic DNA fragments ( ii ) were cloned into the pJAZZ-OK Blunt vector ( i ) to generate the PcG01 and PcG02 genomic libraries. The pJAZZ-OK Blunt vector encode hairpin telomers (shown in black), a telomerase gene (TelN), replication factor and origin (repA), regulator of replication (cB) and a kanamycin resistance gene (kanR). Genomic library clones are converted into gene targeting vectors by recombineering, which introduces a dual bacterial selectable marker zeocin-pheS (zeo-pheS), ( iii ). Linear transfection-ready vectors are then generated by Gateway cloning, which facilitates the exchange of the zeo-pheS cassette for the parasite positive-negative selection marker human dihydrofolate reductase-uridyl phosphoribosyl transferase (hdhfr-yfcu), ( iv ) that are prepared for transfection by NotI digest and integrate with high efficiency into the P. chabaudi genome ( v ).

    Techniques Used: Modification, Clone Assay, Plasmid Preparation, Marker, Transfection, Generated, Selection

    Transfection of P. chabaudi AS to create PcAS-GFP ML . A) Mature P. chabaudi AS schizonts accumulate after 4.5 hours of culture in the presence of C2 (giemsa-stained smear); infected erythrocytes were smeared 5 minutes after C2 was removed from culture. Merozoites undergoing erythrocyte invasion are arrowed. B) Schematic representation of the pCAT 230p -G6 plasmid showing hDHFR and yFcu selectable cassettes, the GFP cassette and 3’PbDHFR-TS direct repeats, allowing recombination and excision of the drug-selectable cassette. C ) Stable integration of GFP into the pc230p locus. To verify correct integration into the pc230p locus in PcAS-GFP.Δ230p DNA, 1.4kb and 1kb fragments were amplified from the 5’ and 3’ integration sites using primers 5’intF x 5’intR and 3’intF x 3’intR, respectively. To verify deletion of pc230p , primers 230pF x 230pR were used to amplify a 0.37kb fragment in wild-type but not PcAS-GFP.Δ230p DNA. To verify the presence of hDHFR, primers dF1 x dF2 were used to amplify a 461bp fragment in PcAS-GFP.Δ230p but not in wild-type or PcAS-GFP ML DNA. D) Recycling of the drug-selectable cassettes. Following selection with 5-FC, recombined parasites excised the drug-selectable cassettes (insert) and an 830bp fragment was amplified from PcAS-GFP ML DNA using primers recF x recR.
    Figure Legend Snippet: Transfection of P. chabaudi AS to create PcAS-GFP ML . A) Mature P. chabaudi AS schizonts accumulate after 4.5 hours of culture in the presence of C2 (giemsa-stained smear); infected erythrocytes were smeared 5 minutes after C2 was removed from culture. Merozoites undergoing erythrocyte invasion are arrowed. B) Schematic representation of the pCAT 230p -G6 plasmid showing hDHFR and yFcu selectable cassettes, the GFP cassette and 3’PbDHFR-TS direct repeats, allowing recombination and excision of the drug-selectable cassette. C ) Stable integration of GFP into the pc230p locus. To verify correct integration into the pc230p locus in PcAS-GFP.Δ230p DNA, 1.4kb and 1kb fragments were amplified from the 5’ and 3’ integration sites using primers 5’intF x 5’intR and 3’intF x 3’intR, respectively. To verify deletion of pc230p , primers 230pF x 230pR were used to amplify a 0.37kb fragment in wild-type but not PcAS-GFP.Δ230p DNA. To verify the presence of hDHFR, primers dF1 x dF2 were used to amplify a 461bp fragment in PcAS-GFP.Δ230p but not in wild-type or PcAS-GFP ML DNA. D) Recycling of the drug-selectable cassettes. Following selection with 5-FC, recombined parasites excised the drug-selectable cassettes (insert) and an 830bp fragment was amplified from PcAS-GFP ML DNA using primers recF x recR.

    Techniques Used: Transfection, Staining, Infection, Plasmid Preparation, Amplification, Selection

    Modification of non-essential loci with pCAT and pJazz plasmids. A) Left hand panel (LHP) ; schematic representation of the pCAT crmp1 -M6 plasmid and the pccrmp1 locus after transfection. Right hand panel (RHP) ; to verify correct integration into the pccrmp1 locus, 1.22kb and 1.17kb fragments were amplified from the 5’ and 3’ integration sites using primers 5’intF.crmp1 x 5’intR.crmp1 and 3’intF.crmp1 x 3’intR.crmp1, respectively. To verify deletion of pccrmp1 , primers crmp1F x crmp1R were used to amplify a 306bp fragment in wild-type but not PcAS-mCh.Δcrmp1 DNA. To verify the presence of hdhfr , primers dF1 x dF2 were used to amplify a 461bp fragment in PcAS-mCh.Δcrmp1 but not in wt DNA. B) LHP ; schematic representation of the PGEM-600068 construct and the pccrmp1 locus after transfection. RHP ; to verify correct integration into the pccrmp1 locus, a 2.9kb fragment was amplified from the 3’ integration site using primers GW1 x GT1.crmp1. To verify deletion of pccrmp1 , primers crmp1F2 x crmp1R2 were used to amplify a 627bp fragment in wt but not PcAS.Δcrmp1 DNA. To verify the presence of hdhfr , primers dF1 x dF2 were used to amplify a 461bp fragment in PcAS.Δcrmp1 but not in wt DNA. C) Day of patency following transfecti o n with pCAT plasmids (n=6) and pJazz constructs (n=8) targeting non-essential loci. Parasitaemia was monitored by giemsa stain from days 7-16 following transfection. Patency was determined when parasitaemia was at least 0.005%. D) LHP ; schematic representation of the PGEM-610754 construct and the pccrmp4 locus after transfection into P. chabaudi AS wild-type and PcAS-GFP ml parasites. RHP ; to verify correct integration into the pccrmp4 locus, a 3.3kb fragment was amplified from the 3’ integration site using primers GW1 x GT1.crmp4. To verify deletion of pccrmp4 in wild-type and PcAS-GFP ml parasites, primers crmp4F x crmp4R were used to amplify a 700bp fragment in wild-type but not PcAS.Δcrmp1 DNA. To verify the presence of hdhfr , primers dF1 x dF2 were used to amplify a 461bp fragment in PcAS.Δcrmp4 but not in wild-type DNA.
    Figure Legend Snippet: Modification of non-essential loci with pCAT and pJazz plasmids. A) Left hand panel (LHP) ; schematic representation of the pCAT crmp1 -M6 plasmid and the pccrmp1 locus after transfection. Right hand panel (RHP) ; to verify correct integration into the pccrmp1 locus, 1.22kb and 1.17kb fragments were amplified from the 5’ and 3’ integration sites using primers 5’intF.crmp1 x 5’intR.crmp1 and 3’intF.crmp1 x 3’intR.crmp1, respectively. To verify deletion of pccrmp1 , primers crmp1F x crmp1R were used to amplify a 306bp fragment in wild-type but not PcAS-mCh.Δcrmp1 DNA. To verify the presence of hdhfr , primers dF1 x dF2 were used to amplify a 461bp fragment in PcAS-mCh.Δcrmp1 but not in wt DNA. B) LHP ; schematic representation of the PGEM-600068 construct and the pccrmp1 locus after transfection. RHP ; to verify correct integration into the pccrmp1 locus, a 2.9kb fragment was amplified from the 3’ integration site using primers GW1 x GT1.crmp1. To verify deletion of pccrmp1 , primers crmp1F2 x crmp1R2 were used to amplify a 627bp fragment in wt but not PcAS.Δcrmp1 DNA. To verify the presence of hdhfr , primers dF1 x dF2 were used to amplify a 461bp fragment in PcAS.Δcrmp1 but not in wt DNA. C) Day of patency following transfecti o n with pCAT plasmids (n=6) and pJazz constructs (n=8) targeting non-essential loci. Parasitaemia was monitored by giemsa stain from days 7-16 following transfection. Patency was determined when parasitaemia was at least 0.005%. D) LHP ; schematic representation of the PGEM-610754 construct and the pccrmp4 locus after transfection into P. chabaudi AS wild-type and PcAS-GFP ml parasites. RHP ; to verify correct integration into the pccrmp4 locus, a 3.3kb fragment was amplified from the 3’ integration site using primers GW1 x GT1.crmp4. To verify deletion of pccrmp4 in wild-type and PcAS-GFP ml parasites, primers crmp4F x crmp4R were used to amplify a 700bp fragment in wild-type but not PcAS.Δcrmp1 DNA. To verify the presence of hdhfr , primers dF1 x dF2 were used to amplify a 461bp fragment in PcAS.Δcrmp4 but not in wild-type DNA.

    Techniques Used: Modification, Plasmid Preparation, Transfection, Amplification, Construct, Giemsa Stain

    6) Product Images from "Identification of a Novel Cleavage Activity of the First Papain-Like Proteinase Domain Encoded by Open Reading Frame 1a of the Coronavirus Avian Infectious Bronchitis Virus and Characterization of the Cleavage Products"

    Article Title: Identification of a Novel Cleavage Activity of the First Papain-Like Proteinase Domain Encoded by Open Reading Frame 1a of the Coronavirus Avian Infectious Bronchitis Virus and Characterization of the Cleavage Products

    Journal: Journal of Virology

    doi:

    (a) Mutational analysis of the putative N-linked glycosylation site of the 41-kDa protein. Plasmids pORF1a4 and pORF1a4N 2313 -Q were transiently expressed in Cos-7 cells, using the vaccinia virus-T7 expression system. Cells were labeled with [ 35 S]methionine, lysates were prepared, and polypeptides were immunoprecipitated with antiserum V46. Gel electrophoresis of the in vivo-synthesized polypeptides (lanes 1 and 2) together with the in vitro-synthesized products (lane 3) was performed on an SDS–12.5% polyacrylamide gel. Polypeptides were detected by fluorography. Numbers indicate molecular masses in kilodaltons. (b) Endo H treatment of the C-terminal cleavage products from expression of pORF1a4. Plasmid DNA was transfected into Cos-7 cells, using the vaccinia virus-T7 expression system. The [ 35 S]methionine-labeled cell lysates were prepared, and polypeptides were immunoprecipitated with antiserum V46. The immunoprecipitated viral proteins were analyzed either by gel electrophoresis directly (lane 4) or after incubation with endo H (40 U/mg; lane 2). Incubation of the immunoprecipitated viral proteins with endo H buffer alone (lane 3) and the 20-kDa cleavage product obtained from in vitro expression of pORF1a4 (lane 1) were included as controls. The [ 35 S]methionine-labeled polypeptides were subjected to gel electrophoresis on an SDS–12.5% polyacrylamide gel, and polypeptides were detected by fluorography. (c) Endo H treatment of the in vitro-synthesized 41-kDa protein. Plasmid pP41 was expressed in vitro, using the cell-free transcription-coupled translation system, in the presence or absence of 5 μl of canine pancreatic microsomal membranes. The [ 35 S]methionine-labeled polypeptides were analyzed either by gel electrophoresis directly or after incubation with endo H (40 U/mg) or buffer only.
    Figure Legend Snippet: (a) Mutational analysis of the putative N-linked glycosylation site of the 41-kDa protein. Plasmids pORF1a4 and pORF1a4N 2313 -Q were transiently expressed in Cos-7 cells, using the vaccinia virus-T7 expression system. Cells were labeled with [ 35 S]methionine, lysates were prepared, and polypeptides were immunoprecipitated with antiserum V46. Gel electrophoresis of the in vivo-synthesized polypeptides (lanes 1 and 2) together with the in vitro-synthesized products (lane 3) was performed on an SDS–12.5% polyacrylamide gel. Polypeptides were detected by fluorography. Numbers indicate molecular masses in kilodaltons. (b) Endo H treatment of the C-terminal cleavage products from expression of pORF1a4. Plasmid DNA was transfected into Cos-7 cells, using the vaccinia virus-T7 expression system. The [ 35 S]methionine-labeled cell lysates were prepared, and polypeptides were immunoprecipitated with antiserum V46. The immunoprecipitated viral proteins were analyzed either by gel electrophoresis directly (lane 4) or after incubation with endo H (40 U/mg; lane 2). Incubation of the immunoprecipitated viral proteins with endo H buffer alone (lane 3) and the 20-kDa cleavage product obtained from in vitro expression of pORF1a4 (lane 1) were included as controls. The [ 35 S]methionine-labeled polypeptides were subjected to gel electrophoresis on an SDS–12.5% polyacrylamide gel, and polypeptides were detected by fluorography. (c) Endo H treatment of the in vitro-synthesized 41-kDa protein. Plasmid pP41 was expressed in vitro, using the cell-free transcription-coupled translation system, in the presence or absence of 5 μl of canine pancreatic microsomal membranes. The [ 35 S]methionine-labeled polypeptides were analyzed either by gel electrophoresis directly or after incubation with endo H (40 U/mg) or buffer only.

    Techniques Used: Expressing, Labeling, Immunoprecipitation, Nucleic Acid Electrophoresis, In Vivo, Synthesized, In Vitro, Plasmid Preparation, Transfection, Incubation

    7) Product Images from "Induction of caspase-dependent apoptosis by betanodaviruses GGNNV and demonstration of protein α as an apoptosis inducer"

    Article Title: Induction of caspase-dependent apoptosis by betanodaviruses GGNNV and demonstration of protein α as an apoptosis inducer

    Journal: Virology

    doi: 10.1016/S0042-6822(02)00098-3

    Protein α induces activation of caspase-3 proteases in both SB and Cos-7 cells. Cell lysates from SB or Cos-7 cells transfected with plasmids pEGFP-RNA2 or pEGFP-C1 were harvested at 24 h post-transfection and assayed for DEVDase activity using the caspase-3 colorimetric substrate DEVD-pNA. In addition, cells transfected with pEGFP-RNA2 were treated with DEVD-CHO. The pEGFP-C1 was used as a negative control and to maintain equal plasmid DNA concentration for each of the transfections. Values shown are means from duplicate experiments.
    Figure Legend Snippet: Protein α induces activation of caspase-3 proteases in both SB and Cos-7 cells. Cell lysates from SB or Cos-7 cells transfected with plasmids pEGFP-RNA2 or pEGFP-C1 were harvested at 24 h post-transfection and assayed for DEVDase activity using the caspase-3 colorimetric substrate DEVD-pNA. In addition, cells transfected with pEGFP-RNA2 were treated with DEVD-CHO. The pEGFP-C1 was used as a negative control and to maintain equal plasmid DNA concentration for each of the transfections. Values shown are means from duplicate experiments.

    Techniques Used: Activation Assay, Transfection, Activity Assay, Negative Control, Plasmid Preparation, Concentration Assay

    (a) Cellular DNA fragmentation induced by transiently expressing protein α in SB and Cos-7 cells. DNA samples were extracted from cells transfected with pEGFP-RNA2 (lane 2) pEGFP-RNA1 (lane 3) and pEGFP-C1 (lane 4) at 48 h post-transfection and DNA fragmentation were detected by 2.0% agarose gel electrophoresis. Lane 1 indicates the DNA size marker. (b) Detection of apoptosis in SB and Cos-7 by TUNEL-labeling. SB or Cos-7 cells were transfected with plasmid pEGFP-RNA2. At 48 h post-transfection, the cells were fixed and analyzed for detection of apoptosis by TUNEL staining, which detects the DNA breakage (brown color). A and C indicate TUNEL-labeling of Cos-7 and SB cells that were transfected with EGFP-RNA2 at 48 h post-transfection, respectively. B and D indicate Cos-7 and SB cells that were transfected with pEGFP-C1 at 48 h post-transfection, respectively. Magnification X200.
    Figure Legend Snippet: (a) Cellular DNA fragmentation induced by transiently expressing protein α in SB and Cos-7 cells. DNA samples were extracted from cells transfected with pEGFP-RNA2 (lane 2) pEGFP-RNA1 (lane 3) and pEGFP-C1 (lane 4) at 48 h post-transfection and DNA fragmentation were detected by 2.0% agarose gel electrophoresis. Lane 1 indicates the DNA size marker. (b) Detection of apoptosis in SB and Cos-7 by TUNEL-labeling. SB or Cos-7 cells were transfected with plasmid pEGFP-RNA2. At 48 h post-transfection, the cells were fixed and analyzed for detection of apoptosis by TUNEL staining, which detects the DNA breakage (brown color). A and C indicate TUNEL-labeling of Cos-7 and SB cells that were transfected with EGFP-RNA2 at 48 h post-transfection, respectively. B and D indicate Cos-7 and SB cells that were transfected with pEGFP-C1 at 48 h post-transfection, respectively. Magnification X200.

    Techniques Used: Expressing, Transfection, Agarose Gel Electrophoresis, Marker, TUNEL Assay, Labeling, Plasmid Preparation, Staining

    8) Product Images from "Development of Cellulosic Secondary Walls in Flax Fibers Requires ?-Galactosidase 1Development of Cellulosic Secondary Walls in Flax Fibers Requires ?-Galactosidase 1 [C]Development of Cellulosic Secondary Walls in Flax Fibers Requires ?-Galactosidase 1 [C] [W]Development of Cellulosic Secondary Walls in Flax Fibers Requires ?-Galactosidase 1 [C] [W] [OA]"

    Article Title: Development of Cellulosic Secondary Walls in Flax Fibers Requires ?-Galactosidase 1Development of Cellulosic Secondary Walls in Flax Fibers Requires ?-Galactosidase 1 [C]Development of Cellulosic Secondary Walls in Flax Fibers Requires ?-Galactosidase 1 [C] [W]Development of Cellulosic Secondary Walls in Flax Fibers Requires ?-Galactosidase 1 [C] [W] [OA]

    Journal: Plant Physiology

    doi: 10.1104/pp.111.172676

    Transcript expression and enzyme activity. A, Relative transcript abundance of LuBGAL1 in three independent LuBGAL-RNAi lines (1–3). Transcript expression is shown relative to a nontransgenic sibling (WT), which is shown with an arbitrary value
    Figure Legend Snippet: Transcript expression and enzyme activity. A, Relative transcript abundance of LuBGAL1 in three independent LuBGAL-RNAi lines (1–3). Transcript expression is shown relative to a nontransgenic sibling (WT), which is shown with an arbitrary value

    Techniques Used: Expressing, Activity Assay

    Stem cross sections immunolabeled with the LM5 antibody. Fluorescent microscopy of nontransgenic (A) and a representative LuBGAL-RNAi line (B). TEM immunogold microscopy of a nontransgenic line (C) and a LuBGAL1-RNAi line (D). Bars = 100 μm in
    Figure Legend Snippet: Stem cross sections immunolabeled with the LM5 antibody. Fluorescent microscopy of nontransgenic (A) and a representative LuBGAL-RNAi line (B). TEM immunogold microscopy of a nontransgenic line (C) and a LuBGAL1-RNAi line (D). Bars = 100 μm in

    Techniques Used: Immunolabeling, Microscopy, Transmission Electron Microscopy

    . For clarity, flax gene identifiers are not shown, except for LuBGAL1 (Lu01) and LuBGAL2 (Lu02)
    Figure Legend Snippet: . For clarity, flax gene identifiers are not shown, except for LuBGAL1 (Lu01) and LuBGAL2 (Lu02)

    Techniques Used:

    Related Articles

    Transfection:

    Article Title: Induction of caspase-dependent apoptosis by betanodaviruses GGNNV and demonstration of protein α as an apoptosis inducer
    Article Snippet: .. In vitro expression of the pEGFP-RNA1 and pEGFP-RNA2 constructs was performed in transient expression experiments using SB and Cos-7 cells, and 60–80% confluent monolayers of SB or Cos-7 cells grown in 25 × 25 cm flask were transfected with a 2 μg/flask of plasmid DNA (purified by Qiagen plasmid Midi kits, Chatsworth, CA, USA) mixed with lipofectamin plus reagent according to the instructions of the manufacturer (Life Technologies, Gaithersburg, MD, USA). .. Western-blot analysis was carried out to determine the expression of protein A and protein α.

    Article Title: Identification of a Novel Cleavage Activity of the First Papain-Like Proteinase Domain Encoded by Open Reading Frame 1a of the Coronavirus Avian Infectious Bronchitis Virus and Characterization of the Cleavage Products
    Article Snippet: .. The cells were then transfected with 2.5 μg of plasmid DNA (purified by Qiagen plasmid Midi kits) mixed with DOTAP liposomal transfection reagent according to the instructions of the manufacturer (Boehringer Mannheim). .. The transfection efficiency achieved by this method was about 25 to 35%, as determined by monitoring the expression of a reporter plasmid encoding green fluorescent protein from jellyfish Aequorea victoria ( ).

    In Vitro:

    Article Title: Induction of caspase-dependent apoptosis by betanodaviruses GGNNV and demonstration of protein α as an apoptosis inducer
    Article Snippet: .. In vitro expression of the pEGFP-RNA1 and pEGFP-RNA2 constructs was performed in transient expression experiments using SB and Cos-7 cells, and 60–80% confluent monolayers of SB or Cos-7 cells grown in 25 × 25 cm flask were transfected with a 2 μg/flask of plasmid DNA (purified by Qiagen plasmid Midi kits, Chatsworth, CA, USA) mixed with lipofectamin plus reagent according to the instructions of the manufacturer (Life Technologies, Gaithersburg, MD, USA). .. Western-blot analysis was carried out to determine the expression of protein A and protein α.

    Labeling:

    Article Title: Genomic Diversity in Campylobacter jejuni: Identification of C. jejuni 81-176-Specific Genes
    Article Snippet: .. The plasmids were extracted from C. jejuni 81-176 using the QIAGEN plasmid Midi kit, labeled with the Cy3 fluorescent dye, and cohybridized on the shotgun array with Cy5-labeled genomic DNA. .. Spots with a signal intensity above 2 times the standard deviation of the background in the Cy3 channel were selected as containing DNA fragments belonging to one of the two plasmids.

    Purification:

    Article Title: Structure of trigger factor binding domain in biologically homologous complex with eubacterial ribosome reveals its chaperone action
    Article Snippet: .. The bacteria were grown, and the plasmids were purified by using a Plasmid midi kit (Qiagen). ..

    Article Title: Structural Alteration of OmpR as a Source of Ertapenem Resistance in a CTX-M-15-Producing Escherichia coli O25b:H4 Sequence Type 131 Clinical Isolate
    Article Snippet: .. The recombinant plasmid was purified using the QIAquick plasmid midi kit (Qiagen) and transformed into E. coli strain ErtR, as described previously ( ). .. OMPs were isolated according to the rapid procedure of Carlone et al. ( ) and separated by SDS-PAGE, as previously described ( ).

    Article Title: Induction of caspase-dependent apoptosis by betanodaviruses GGNNV and demonstration of protein α as an apoptosis inducer
    Article Snippet: .. In vitro expression of the pEGFP-RNA1 and pEGFP-RNA2 constructs was performed in transient expression experiments using SB and Cos-7 cells, and 60–80% confluent monolayers of SB or Cos-7 cells grown in 25 × 25 cm flask were transfected with a 2 μg/flask of plasmid DNA (purified by Qiagen plasmid Midi kits, Chatsworth, CA, USA) mixed with lipofectamin plus reagent according to the instructions of the manufacturer (Life Technologies, Gaithersburg, MD, USA). .. Western-blot analysis was carried out to determine the expression of protein A and protein α.

    Article Title: Identification of a Novel Cleavage Activity of the First Papain-Like Proteinase Domain Encoded by Open Reading Frame 1a of the Coronavirus Avian Infectious Bronchitis Virus and Characterization of the Cleavage Products
    Article Snippet: .. The cells were then transfected with 2.5 μg of plasmid DNA (purified by Qiagen plasmid Midi kits) mixed with DOTAP liposomal transfection reagent according to the instructions of the manufacturer (Boehringer Mannheim). .. The transfection efficiency achieved by this method was about 25 to 35%, as determined by monitoring the expression of a reporter plasmid encoding green fluorescent protein from jellyfish Aequorea victoria ( ).

    Article Title: The Stress-Inducible Peroxidase TSA2 Underlies a Conditionally Beneficial Chromosomal Duplication in Saccharomyces cerevisiae
    Article Snippet: .. Plasmids were purified from transformed strains using the Qiagen midi plasmid purification kit and transformed into euploid progenitor strains. .. Based on Sanger sequencing, the cloned TSA2 locus was found to have the same sequence as the BY × RM progenitor.

    Immunoprecipitation:

    Article Title: MID1 and MID2 homo- and heterodimerise to tether the rapamycin-sensitive PP2A regulatory subunit, Alpha 4, to microtubules: implications for the clinical variability of X-linked Opitz GBBB syndrome and other developmental disorders
    Article Snippet: .. Immunoprecipitation and western analysis Preparations of the various GFP- and myc-tagged expression constructs were made using a DNA plasmid Midi kit (Qiagen). .. Six picomoles (approximately 3 micrograms) of each construct were transfected into approximately 107 Cos1 cells using FuGene transfection reagent (Roche Diagnostics Australia).

    Construct:

    Article Title: Induction of caspase-dependent apoptosis by betanodaviruses GGNNV and demonstration of protein α as an apoptosis inducer
    Article Snippet: .. In vitro expression of the pEGFP-RNA1 and pEGFP-RNA2 constructs was performed in transient expression experiments using SB and Cos-7 cells, and 60–80% confluent monolayers of SB or Cos-7 cells grown in 25 × 25 cm flask were transfected with a 2 μg/flask of plasmid DNA (purified by Qiagen plasmid Midi kits, Chatsworth, CA, USA) mixed with lipofectamin plus reagent according to the instructions of the manufacturer (Life Technologies, Gaithersburg, MD, USA). .. Western-blot analysis was carried out to determine the expression of protein A and protein α.

    Article Title: MID1 and MID2 homo- and heterodimerise to tether the rapamycin-sensitive PP2A regulatory subunit, Alpha 4, to microtubules: implications for the clinical variability of X-linked Opitz GBBB syndrome and other developmental disorders
    Article Snippet: .. Immunoprecipitation and western analysis Preparations of the various GFP- and myc-tagged expression constructs were made using a DNA plasmid Midi kit (Qiagen). .. Six picomoles (approximately 3 micrograms) of each construct were transfected into approximately 107 Cos1 cells using FuGene transfection reagent (Roche Diagnostics Australia).

    Expressing:

    Article Title: Induction of caspase-dependent apoptosis by betanodaviruses GGNNV and demonstration of protein α as an apoptosis inducer
    Article Snippet: .. In vitro expression of the pEGFP-RNA1 and pEGFP-RNA2 constructs was performed in transient expression experiments using SB and Cos-7 cells, and 60–80% confluent monolayers of SB or Cos-7 cells grown in 25 × 25 cm flask were transfected with a 2 μg/flask of plasmid DNA (purified by Qiagen plasmid Midi kits, Chatsworth, CA, USA) mixed with lipofectamin plus reagent according to the instructions of the manufacturer (Life Technologies, Gaithersburg, MD, USA). .. Western-blot analysis was carried out to determine the expression of protein A and protein α.

    Article Title: MID1 and MID2 homo- and heterodimerise to tether the rapamycin-sensitive PP2A regulatory subunit, Alpha 4, to microtubules: implications for the clinical variability of X-linked Opitz GBBB syndrome and other developmental disorders
    Article Snippet: .. Immunoprecipitation and western analysis Preparations of the various GFP- and myc-tagged expression constructs were made using a DNA plasmid Midi kit (Qiagen). .. Six picomoles (approximately 3 micrograms) of each construct were transfected into approximately 107 Cos1 cells using FuGene transfection reagent (Roche Diagnostics Australia).

    Western Blot:

    Article Title: MID1 and MID2 homo- and heterodimerise to tether the rapamycin-sensitive PP2A regulatory subunit, Alpha 4, to microtubules: implications for the clinical variability of X-linked Opitz GBBB syndrome and other developmental disorders
    Article Snippet: .. Immunoprecipitation and western analysis Preparations of the various GFP- and myc-tagged expression constructs were made using a DNA plasmid Midi kit (Qiagen). .. Six picomoles (approximately 3 micrograms) of each construct were transfected into approximately 107 Cos1 cells using FuGene transfection reagent (Roche Diagnostics Australia).

    Transformation Assay:

    Article Title: Structural Alteration of OmpR as a Source of Ertapenem Resistance in a CTX-M-15-Producing Escherichia coli O25b:H4 Sequence Type 131 Clinical Isolate
    Article Snippet: .. The recombinant plasmid was purified using the QIAquick plasmid midi kit (Qiagen) and transformed into E. coli strain ErtR, as described previously ( ). .. OMPs were isolated according to the rapid procedure of Carlone et al. ( ) and separated by SDS-PAGE, as previously described ( ).

    Article Title: The Stress-Inducible Peroxidase TSA2 Underlies a Conditionally Beneficial Chromosomal Duplication in Saccharomyces cerevisiae
    Article Snippet: .. Plasmids were purified from transformed strains using the Qiagen midi plasmid purification kit and transformed into euploid progenitor strains. .. Based on Sanger sequencing, the cloned TSA2 locus was found to have the same sequence as the BY × RM progenitor.

    Recombinant:

    Article Title: Structural Alteration of OmpR as a Source of Ertapenem Resistance in a CTX-M-15-Producing Escherichia coli O25b:H4 Sequence Type 131 Clinical Isolate
    Article Snippet: .. The recombinant plasmid was purified using the QIAquick plasmid midi kit (Qiagen) and transformed into E. coli strain ErtR, as described previously ( ). .. OMPs were isolated according to the rapid procedure of Carlone et al. ( ) and separated by SDS-PAGE, as previously described ( ).

    Plasmid Preparation:

    Article Title: Structure of trigger factor binding domain in biologically homologous complex with eubacterial ribosome reveals its chaperone action
    Article Snippet: .. The bacteria were grown, and the plasmids were purified by using a Plasmid midi kit (Qiagen). ..

    Article Title: Integron Carrying a Novel Metallo-?-Lactamase Gene, blaIMP-16, and a Fused Form of Aminoglycoside-Resistant Gene aac(6′)-30/aac(6′)-Ib′: Report from the SENTRY Antimicrobial Surveillance Program
    Article Snippet: .. Plasmid DNA from P. aeruginosa 101-4704 was extracted with a plasmid DNA Midi kit (Qiagen, Chatsworth, Calif.). .. The transfer of β-lactam resistance markers from strain 101-4704 into E. coli DH5α and a rifampin-resistant (Rifr ) mutant of P. aeruginosa pA01 was performed with a Gene Pulser apparatus (Bio-Rad, Watford, United Kingdom) that was set at 2.5 kV, 25 μF, and 400 Ω.

    Article Title: Structural Alteration of OmpR as a Source of Ertapenem Resistance in a CTX-M-15-Producing Escherichia coli O25b:H4 Sequence Type 131 Clinical Isolate
    Article Snippet: .. The recombinant plasmid was purified using the QIAquick plasmid midi kit (Qiagen) and transformed into E. coli strain ErtR, as described previously ( ). .. OMPs were isolated according to the rapid procedure of Carlone et al. ( ) and separated by SDS-PAGE, as previously described ( ).

    Article Title: Induction of caspase-dependent apoptosis by betanodaviruses GGNNV and demonstration of protein α as an apoptosis inducer
    Article Snippet: .. In vitro expression of the pEGFP-RNA1 and pEGFP-RNA2 constructs was performed in transient expression experiments using SB and Cos-7 cells, and 60–80% confluent monolayers of SB or Cos-7 cells grown in 25 × 25 cm flask were transfected with a 2 μg/flask of plasmid DNA (purified by Qiagen plasmid Midi kits, Chatsworth, CA, USA) mixed with lipofectamin plus reagent according to the instructions of the manufacturer (Life Technologies, Gaithersburg, MD, USA). .. Western-blot analysis was carried out to determine the expression of protein A and protein α.

    Article Title: MID1 and MID2 homo- and heterodimerise to tether the rapamycin-sensitive PP2A regulatory subunit, Alpha 4, to microtubules: implications for the clinical variability of X-linked Opitz GBBB syndrome and other developmental disorders
    Article Snippet: .. Immunoprecipitation and western analysis Preparations of the various GFP- and myc-tagged expression constructs were made using a DNA plasmid Midi kit (Qiagen). .. Six picomoles (approximately 3 micrograms) of each construct were transfected into approximately 107 Cos1 cells using FuGene transfection reagent (Roche Diagnostics Australia).

    Article Title: Genomic Diversity in Campylobacter jejuni: Identification of C. jejuni 81-176-Specific Genes
    Article Snippet: .. The plasmids were extracted from C. jejuni 81-176 using the QIAGEN plasmid Midi kit, labeled with the Cy3 fluorescent dye, and cohybridized on the shotgun array with Cy5-labeled genomic DNA. .. Spots with a signal intensity above 2 times the standard deviation of the background in the Cy3 channel were selected as containing DNA fragments belonging to one of the two plasmids.

    Article Title: Identification of a Novel Cleavage Activity of the First Papain-Like Proteinase Domain Encoded by Open Reading Frame 1a of the Coronavirus Avian Infectious Bronchitis Virus and Characterization of the Cleavage Products
    Article Snippet: .. The cells were then transfected with 2.5 μg of plasmid DNA (purified by Qiagen plasmid Midi kits) mixed with DOTAP liposomal transfection reagent according to the instructions of the manufacturer (Boehringer Mannheim). .. The transfection efficiency achieved by this method was about 25 to 35%, as determined by monitoring the expression of a reporter plasmid encoding green fluorescent protein from jellyfish Aequorea victoria ( ).

    Article Title: The Stress-Inducible Peroxidase TSA2 Underlies a Conditionally Beneficial Chromosomal Duplication in Saccharomyces cerevisiae
    Article Snippet: .. Plasmids were purified from transformed strains using the Qiagen midi plasmid purification kit and transformed into euploid progenitor strains. .. Based on Sanger sequencing, the cloned TSA2 locus was found to have the same sequence as the BY × RM progenitor.

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    Qiagen plasmid dna midi kit
    Schematic representation of the class 1 integron-containing bla IMP-16 gene cassette from clinical isolate P. <t>aeruginosa</t> 101-4704. Boxes, inserted genes; arrows, transcriptional orientations; black circles, 59-be's; white circle, attI1 recombination site; lines, <t>DNA</t> of the inserts contained within recombinant plasmids pREM-1, pREM-2, pREM-3, and pREM-4; arrowheads, primer positions and their orientations; M, start codon; asterisk, the location of the stop codon for the particular gene.
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    Schematic representation of the class 1 integron-containing bla IMP-16 gene cassette from clinical isolate P. aeruginosa 101-4704. Boxes, inserted genes; arrows, transcriptional orientations; black circles, 59-be's; white circle, attI1 recombination site; lines, DNA of the inserts contained within recombinant plasmids pREM-1, pREM-2, pREM-3, and pREM-4; arrowheads, primer positions and their orientations; M, start codon; asterisk, the location of the stop codon for the particular gene.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Integron Carrying a Novel Metallo-?-Lactamase Gene, blaIMP-16, and a Fused Form of Aminoglycoside-Resistant Gene aac(6′)-30/aac(6′)-Ib′: Report from the SENTRY Antimicrobial Surveillance Program

    doi: 10.1128/AAC.48.12.4693-4702.2004

    Figure Lengend Snippet: Schematic representation of the class 1 integron-containing bla IMP-16 gene cassette from clinical isolate P. aeruginosa 101-4704. Boxes, inserted genes; arrows, transcriptional orientations; black circles, 59-be's; white circle, attI1 recombination site; lines, DNA of the inserts contained within recombinant plasmids pREM-1, pREM-2, pREM-3, and pREM-4; arrowheads, primer positions and their orientations; M, start codon; asterisk, the location of the stop codon for the particular gene.

    Article Snippet: Plasmid DNA from P. aeruginosa 101-4704 was extracted with a plasmid DNA Midi kit (Qiagen, Chatsworth, Calif.).

    Techniques: Recombinant

    Alpha 4 interacts with MID1 and MID2. (A) Yeast two-hybrid analysis of the interaction between MID1 and Alpha 4 as well as MID2 and Alpha 4. Yeast agar plate (leu - trp - his - , 75 mM 3-AT) showing growth for MID1/Alpha 4 and MID2/Alpha 4 interactions as well as positive control two-hybrid combinations and no growth for negative controls. (B) Detection of full-length myc tagged-Alpha 4 when co-expressed with GFP-MID1 and GFP-MID2 fusion proteins in transiently transfected Cos1 cells. (a) GFP-MID1 fluorescence (green), (b) anti-myc antibody detecting myc-Alpha 4 localisation (red), (c) overlay of (a), (b) showing co-localisation of the myc-Alpha 4 fusion protein and GFP-MID1, with DAPI stain for DNA (blue). (d) GFP-MID2 fluorescence (green), (e) myc-Alpha 4 localisation (using same detection as b) (red), (f) overlay of (d), (e) with DAPI (blue) showing co-localisation of the myc-Alpha 4 fusion protein and GFP-MID2. (g) Detection of transiently expressed myc-Alpha 4 fusion protein in Cos1 cells, (h) overlay of (g) and DAPI stain showing cytoplasmic distribution of myc-Alpha 4 fusion protein.

    Journal: BMC Cell Biology

    Article Title: MID1 and MID2 homo- and heterodimerise to tether the rapamycin-sensitive PP2A regulatory subunit, Alpha 4, to microtubules: implications for the clinical variability of X-linked Opitz GBBB syndrome and other developmental disorders

    doi: 10.1186/1471-2121-3-1

    Figure Lengend Snippet: Alpha 4 interacts with MID1 and MID2. (A) Yeast two-hybrid analysis of the interaction between MID1 and Alpha 4 as well as MID2 and Alpha 4. Yeast agar plate (leu - trp - his - , 75 mM 3-AT) showing growth for MID1/Alpha 4 and MID2/Alpha 4 interactions as well as positive control two-hybrid combinations and no growth for negative controls. (B) Detection of full-length myc tagged-Alpha 4 when co-expressed with GFP-MID1 and GFP-MID2 fusion proteins in transiently transfected Cos1 cells. (a) GFP-MID1 fluorescence (green), (b) anti-myc antibody detecting myc-Alpha 4 localisation (red), (c) overlay of (a), (b) showing co-localisation of the myc-Alpha 4 fusion protein and GFP-MID1, with DAPI stain for DNA (blue). (d) GFP-MID2 fluorescence (green), (e) myc-Alpha 4 localisation (using same detection as b) (red), (f) overlay of (d), (e) with DAPI (blue) showing co-localisation of the myc-Alpha 4 fusion protein and GFP-MID2. (g) Detection of transiently expressed myc-Alpha 4 fusion protein in Cos1 cells, (h) overlay of (g) and DAPI stain showing cytoplasmic distribution of myc-Alpha 4 fusion protein.

    Article Snippet: Immunoprecipitation and western analysis Preparations of the various GFP- and myc-tagged expression constructs were made using a DNA plasmid Midi kit (Qiagen).

    Techniques: Positive Control, Transfection, Fluorescence, Staining

    (a) Mutational analysis of the putative N-linked glycosylation site of the 41-kDa protein. Plasmids pORF1a4 and pORF1a4N 2313 -Q were transiently expressed in Cos-7 cells, using the vaccinia virus-T7 expression system. Cells were labeled with [ 35 S]methionine, lysates were prepared, and polypeptides were immunoprecipitated with antiserum V46. Gel electrophoresis of the in vivo-synthesized polypeptides (lanes 1 and 2) together with the in vitro-synthesized products (lane 3) was performed on an SDS–12.5% polyacrylamide gel. Polypeptides were detected by fluorography. Numbers indicate molecular masses in kilodaltons. (b) Endo H treatment of the C-terminal cleavage products from expression of pORF1a4. Plasmid DNA was transfected into Cos-7 cells, using the vaccinia virus-T7 expression system. The [ 35 S]methionine-labeled cell lysates were prepared, and polypeptides were immunoprecipitated with antiserum V46. The immunoprecipitated viral proteins were analyzed either by gel electrophoresis directly (lane 4) or after incubation with endo H (40 U/mg; lane 2). Incubation of the immunoprecipitated viral proteins with endo H buffer alone (lane 3) and the 20-kDa cleavage product obtained from in vitro expression of pORF1a4 (lane 1) were included as controls. The [ 35 S]methionine-labeled polypeptides were subjected to gel electrophoresis on an SDS–12.5% polyacrylamide gel, and polypeptides were detected by fluorography. (c) Endo H treatment of the in vitro-synthesized 41-kDa protein. Plasmid pP41 was expressed in vitro, using the cell-free transcription-coupled translation system, in the presence or absence of 5 μl of canine pancreatic microsomal membranes. The [ 35 S]methionine-labeled polypeptides were analyzed either by gel electrophoresis directly or after incubation with endo H (40 U/mg) or buffer only.

    Journal: Journal of Virology

    Article Title: Identification of a Novel Cleavage Activity of the First Papain-Like Proteinase Domain Encoded by Open Reading Frame 1a of the Coronavirus Avian Infectious Bronchitis Virus and Characterization of the Cleavage Products

    doi:

    Figure Lengend Snippet: (a) Mutational analysis of the putative N-linked glycosylation site of the 41-kDa protein. Plasmids pORF1a4 and pORF1a4N 2313 -Q were transiently expressed in Cos-7 cells, using the vaccinia virus-T7 expression system. Cells were labeled with [ 35 S]methionine, lysates were prepared, and polypeptides were immunoprecipitated with antiserum V46. Gel electrophoresis of the in vivo-synthesized polypeptides (lanes 1 and 2) together with the in vitro-synthesized products (lane 3) was performed on an SDS–12.5% polyacrylamide gel. Polypeptides were detected by fluorography. Numbers indicate molecular masses in kilodaltons. (b) Endo H treatment of the C-terminal cleavage products from expression of pORF1a4. Plasmid DNA was transfected into Cos-7 cells, using the vaccinia virus-T7 expression system. The [ 35 S]methionine-labeled cell lysates were prepared, and polypeptides were immunoprecipitated with antiserum V46. The immunoprecipitated viral proteins were analyzed either by gel electrophoresis directly (lane 4) or after incubation with endo H (40 U/mg; lane 2). Incubation of the immunoprecipitated viral proteins with endo H buffer alone (lane 3) and the 20-kDa cleavage product obtained from in vitro expression of pORF1a4 (lane 1) were included as controls. The [ 35 S]methionine-labeled polypeptides were subjected to gel electrophoresis on an SDS–12.5% polyacrylamide gel, and polypeptides were detected by fluorography. (c) Endo H treatment of the in vitro-synthesized 41-kDa protein. Plasmid pP41 was expressed in vitro, using the cell-free transcription-coupled translation system, in the presence or absence of 5 μl of canine pancreatic microsomal membranes. The [ 35 S]methionine-labeled polypeptides were analyzed either by gel electrophoresis directly or after incubation with endo H (40 U/mg) or buffer only.

    Article Snippet: The cells were then transfected with 2.5 μg of plasmid DNA (purified by Qiagen plasmid Midi kits) mixed with DOTAP liposomal transfection reagent according to the instructions of the manufacturer (Boehringer Mannheim).

    Techniques: Expressing, Labeling, Immunoprecipitation, Nucleic Acid Electrophoresis, In Vivo, Synthesized, In Vitro, Plasmid Preparation, Transfection, Incubation

    Protein α induces activation of caspase-3 proteases in both SB and Cos-7 cells. Cell lysates from SB or Cos-7 cells transfected with plasmids pEGFP-RNA2 or pEGFP-C1 were harvested at 24 h post-transfection and assayed for DEVDase activity using the caspase-3 colorimetric substrate DEVD-pNA. In addition, cells transfected with pEGFP-RNA2 were treated with DEVD-CHO. The pEGFP-C1 was used as a negative control and to maintain equal plasmid DNA concentration for each of the transfections. Values shown are means from duplicate experiments.

    Journal: Virology

    Article Title: Induction of caspase-dependent apoptosis by betanodaviruses GGNNV and demonstration of protein α as an apoptosis inducer

    doi: 10.1016/S0042-6822(02)00098-3

    Figure Lengend Snippet: Protein α induces activation of caspase-3 proteases in both SB and Cos-7 cells. Cell lysates from SB or Cos-7 cells transfected with plasmids pEGFP-RNA2 or pEGFP-C1 were harvested at 24 h post-transfection and assayed for DEVDase activity using the caspase-3 colorimetric substrate DEVD-pNA. In addition, cells transfected with pEGFP-RNA2 were treated with DEVD-CHO. The pEGFP-C1 was used as a negative control and to maintain equal plasmid DNA concentration for each of the transfections. Values shown are means from duplicate experiments.

    Article Snippet: In vitro expression of the pEGFP-RNA1 and pEGFP-RNA2 constructs was performed in transient expression experiments using SB and Cos-7 cells, and 60–80% confluent monolayers of SB or Cos-7 cells grown in 25 × 25 cm flask were transfected with a 2 μg/flask of plasmid DNA (purified by Qiagen plasmid Midi kits, Chatsworth, CA, USA) mixed with lipofectamin plus reagent according to the instructions of the manufacturer (Life Technologies, Gaithersburg, MD, USA).

    Techniques: Activation Assay, Transfection, Activity Assay, Negative Control, Plasmid Preparation, Concentration Assay

    (a) Cellular DNA fragmentation induced by transiently expressing protein α in SB and Cos-7 cells. DNA samples were extracted from cells transfected with pEGFP-RNA2 (lane 2) pEGFP-RNA1 (lane 3) and pEGFP-C1 (lane 4) at 48 h post-transfection and DNA fragmentation were detected by 2.0% agarose gel electrophoresis. Lane 1 indicates the DNA size marker. (b) Detection of apoptosis in SB and Cos-7 by TUNEL-labeling. SB or Cos-7 cells were transfected with plasmid pEGFP-RNA2. At 48 h post-transfection, the cells were fixed and analyzed for detection of apoptosis by TUNEL staining, which detects the DNA breakage (brown color). A and C indicate TUNEL-labeling of Cos-7 and SB cells that were transfected with EGFP-RNA2 at 48 h post-transfection, respectively. B and D indicate Cos-7 and SB cells that were transfected with pEGFP-C1 at 48 h post-transfection, respectively. Magnification X200.

    Journal: Virology

    Article Title: Induction of caspase-dependent apoptosis by betanodaviruses GGNNV and demonstration of protein α as an apoptosis inducer

    doi: 10.1016/S0042-6822(02)00098-3

    Figure Lengend Snippet: (a) Cellular DNA fragmentation induced by transiently expressing protein α in SB and Cos-7 cells. DNA samples were extracted from cells transfected with pEGFP-RNA2 (lane 2) pEGFP-RNA1 (lane 3) and pEGFP-C1 (lane 4) at 48 h post-transfection and DNA fragmentation were detected by 2.0% agarose gel electrophoresis. Lane 1 indicates the DNA size marker. (b) Detection of apoptosis in SB and Cos-7 by TUNEL-labeling. SB or Cos-7 cells were transfected with plasmid pEGFP-RNA2. At 48 h post-transfection, the cells were fixed and analyzed for detection of apoptosis by TUNEL staining, which detects the DNA breakage (brown color). A and C indicate TUNEL-labeling of Cos-7 and SB cells that were transfected with EGFP-RNA2 at 48 h post-transfection, respectively. B and D indicate Cos-7 and SB cells that were transfected with pEGFP-C1 at 48 h post-transfection, respectively. Magnification X200.

    Article Snippet: In vitro expression of the pEGFP-RNA1 and pEGFP-RNA2 constructs was performed in transient expression experiments using SB and Cos-7 cells, and 60–80% confluent monolayers of SB or Cos-7 cells grown in 25 × 25 cm flask were transfected with a 2 μg/flask of plasmid DNA (purified by Qiagen plasmid Midi kits, Chatsworth, CA, USA) mixed with lipofectamin plus reagent according to the instructions of the manufacturer (Life Technologies, Gaithersburg, MD, USA).

    Techniques: Expressing, Transfection, Agarose Gel Electrophoresis, Marker, TUNEL Assay, Labeling, Plasmid Preparation, Staining