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  • 99
    Millipore ha83 protein expression vectors
    Subcellular localization of <t>Ha83</t> and truncated mutants as demonstrated by confocal microscopy. HzAM1 cells were co-transfected with HaBacHZ8 bacmid DNA and pGE-Ha83, pGE-TC151, pGE-TC142, pGE-TC128, pGE-TC118, pGE-TC113, and pGE-TC71, respectively. At the designated time points, cells were fixed, inmmunostained with TRITC-labeled phalloidine to stain the cytoskeleton protein (red) and 4′,6-diamidino-2-phenylindole (Dapi) to stain the nucleus (blue). Mock cells were used as a control. ( A ) Subcellular localization of Ha83 and truncated mutants at 24 h p.t. ( B ) Subcellular localization of Ha83 and truncated mutants at 48 h p.t.
    Ha83 Protein Expression Vectors, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore pet 28a
    SDS-PAGE and Western blot analysis of the expression of recombinant Ifu-Chit2 in E. coli . Lane M: pre-stained protein marker; Lane 1: total protein of cells containing empty vector <t>pET-28a</t> with IPTG induction; Lane 2: total protein of cells containing expression vector pET- 28a-Ifu-chit2 without IPTG induction; Lanes 3–8: total protein of cells containing expression vector pET- 28a-Ifu-chit2 induced by IPTG at 28 °C for 1, 2, 3, 4, 5 and 6 h, respectively; Lane 9: purified recombinant Ifu-Chit2 (50 kDa). Lane 10: total protein of cells containing empty vector pET-28a with IPTG induction; Lane 11: total protein of cells containing expression vector pET- 28a-Ifu-chit2 without IPTG induction; Lane 12: total protein of cells containing expression vector pET- 28a-Ifu-chit2 induced by IPTG at 28 °C for 4 h; Lane13: Purified recombinant Ifu-Chit2.
    Pet 28a, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 8609 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore pet ­28a vector
    SDS-PAGE and Western blot analysis of the expression of recombinant Ifu-Chit2 in E. coli . Lane M: pre-stained protein marker; Lane 1: total protein of cells containing empty vector <t>pET-28a</t> with IPTG induction; Lane 2: total protein of cells containing expression vector pET- 28a-Ifu-chit2 without IPTG induction; Lanes 3–8: total protein of cells containing expression vector pET- 28a-Ifu-chit2 induced by IPTG at 28 °C for 1, 2, 3, 4, 5 and 6 h, respectively; Lane 9: purified recombinant Ifu-Chit2 (50 kDa). Lane 10: total protein of cells containing empty vector pET-28a with IPTG induction; Lane 11: total protein of cells containing expression vector pET- 28a-Ifu-chit2 without IPTG induction; Lane 12: total protein of cells containing expression vector pET- 28a-Ifu-chit2 induced by IPTG at 28 °C for 4 h; Lane13: Purified recombinant Ifu-Chit2.
    Pet ­28a Vector, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA pet 28a
    SDS-PAGE and Western blot analysis of the expression of recombinant Ifu-Chit2 in E. coli . Lane M: pre-stained protein marker; Lane 1: total protein of cells containing empty vector <t>pET-28a</t> with IPTG induction; Lane 2: total protein of cells containing expression vector pET- 28a-Ifu-chit2 without IPTG induction; Lanes 3–8: total protein of cells containing expression vector pET- 28a-Ifu-chit2 induced by IPTG at 28 °C for 1, 2, 3, 4, 5 and 6 h, respectively; Lane 9: purified recombinant Ifu-Chit2 (50 kDa). Lane 10: total protein of cells containing empty vector pET-28a with IPTG induction; Lane 11: total protein of cells containing expression vector pET- 28a-Ifu-chit2 without IPTG induction; Lane 12: total protein of cells containing expression vector pET- 28a-Ifu-chit2 induced by IPTG at 28 °C for 4 h; Lane13: Purified recombinant Ifu-Chit2.
    Pet 28a, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 238 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore t7 expression vector pet ­28a
    SDS-PAGE and Western blot analysis of the expression of recombinant Ifu-Chit2 in E. coli . Lane M: pre-stained protein marker; Lane 1: total protein of cells containing empty vector <t>pET-28a</t> with IPTG induction; Lane 2: total protein of cells containing expression vector pET- 28a-Ifu-chit2 without IPTG induction; Lanes 3–8: total protein of cells containing expression vector pET- 28a-Ifu-chit2 induced by IPTG at 28 °C for 1, 2, 3, 4, 5 and 6 h, respectively; Lane 9: purified recombinant Ifu-Chit2 (50 kDa). Lane 10: total protein of cells containing empty vector pET-28a with IPTG induction; Lane 11: total protein of cells containing expression vector pET- 28a-Ifu-chit2 without IPTG induction; Lane 12: total protein of cells containing expression vector pET- 28a-Ifu-chit2 induced by IPTG at 28 °C for 4 h; Lane13: Purified recombinant Ifu-Chit2.
    T7 Expression Vector Pet ­28a, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    t7 expression vector pet ­28a - by Bioz Stars, 2020-08
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    85
    Millipore plasmid pet 28a3dpol
    SDS-PAGE and Western blot analysis of the expression of recombinant Ifu-Chit2 in E. coli . Lane M: pre-stained protein marker; Lane 1: total protein of cells containing empty vector <t>pET-28a</t> with IPTG induction; Lane 2: total protein of cells containing expression vector pET- 28a-Ifu-chit2 without IPTG induction; Lanes 3–8: total protein of cells containing expression vector pET- 28a-Ifu-chit2 induced by IPTG at 28 °C for 1, 2, 3, 4, 5 and 6 h, respectively; Lane 9: purified recombinant Ifu-Chit2 (50 kDa). Lane 10: total protein of cells containing empty vector pET-28a with IPTG induction; Lane 11: total protein of cells containing expression vector pET- 28a-Ifu-chit2 without IPTG induction; Lane 12: total protein of cells containing expression vector pET- 28a-Ifu-chit2 induced by IPTG at 28 °C for 4 h; Lane13: Purified recombinant Ifu-Chit2.
    Plasmid Pet 28a3dpol, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore pet
    SDS-PAGE and Western blot analysis of the expression of recombinant Ifu-Chit2 in E. coli . Lane M: pre-stained protein marker; Lane 1: total protein of cells containing empty vector <t>pET-28a</t> with IPTG induction; Lane 2: total protein of cells containing expression vector pET- 28a-Ifu-chit2 without IPTG induction; Lanes 3–8: total protein of cells containing expression vector pET- 28a-Ifu-chit2 induced by IPTG at 28 °C for 1, 2, 3, 4, 5 and 6 h, respectively; Lane 9: purified recombinant Ifu-Chit2 (50 kDa). Lane 10: total protein of cells containing empty vector pET-28a with IPTG induction; Lane 11: total protein of cells containing expression vector pET- 28a-Ifu-chit2 without IPTG induction; Lane 12: total protein of cells containing expression vector pET- 28a-Ifu-chit2 induced by IPTG at 28 °C for 4 h; Lane13: Purified recombinant Ifu-Chit2.
    Pet, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 312 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore novagen s pet 28a vector
    Agarose gel showing confirmation of insert in pET <t>28a.</t> lane 1 is 100 bp DNA ladder, lane 2 is uncut pET 28a-Ndk construct and lane 3 is restriction digestion of pET 28a-Ndk gene.
    Novagen S Pet 28a Vector, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 512 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore ecori xhoi digested pet 28a
    Agarose gel showing confirmation of insert in pET <t>28a.</t> lane 1 is 100 bp DNA ladder, lane 2 is uncut pET 28a-Ndk construct and lane 3 is restriction digestion of pET 28a-Ndk gene.
    Ecori Xhoi Digested Pet 28a, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Subcellular localization of Ha83 and truncated mutants as demonstrated by confocal microscopy. HzAM1 cells were co-transfected with HaBacHZ8 bacmid DNA and pGE-Ha83, pGE-TC151, pGE-TC142, pGE-TC128, pGE-TC118, pGE-TC113, and pGE-TC71, respectively. At the designated time points, cells were fixed, inmmunostained with TRITC-labeled phalloidine to stain the cytoskeleton protein (red) and 4′,6-diamidino-2-phenylindole (Dapi) to stain the nucleus (blue). Mock cells were used as a control. ( A ) Subcellular localization of Ha83 and truncated mutants at 24 h p.t. ( B ) Subcellular localization of Ha83 and truncated mutants at 48 h p.t.

    Journal: Scientific Reports

    Article Title: Ha83, a Chitin Binding Domain Encoding Gene, Is Important to Helicoverpa armigera Nucleopolyhedrovirus Budded Virus Production and Occlusion Body Assembling

    doi: 10.1038/srep11088

    Figure Lengend Snippet: Subcellular localization of Ha83 and truncated mutants as demonstrated by confocal microscopy. HzAM1 cells were co-transfected with HaBacHZ8 bacmid DNA and pGE-Ha83, pGE-TC151, pGE-TC142, pGE-TC128, pGE-TC118, pGE-TC113, and pGE-TC71, respectively. At the designated time points, cells were fixed, inmmunostained with TRITC-labeled phalloidine to stain the cytoskeleton protein (red) and 4′,6-diamidino-2-phenylindole (Dapi) to stain the nucleus (blue). Mock cells were used as a control. ( A ) Subcellular localization of Ha83 and truncated mutants at 24 h p.t. ( B ) Subcellular localization of Ha83 and truncated mutants at 48 h p.t.

    Article Snippet: Six truncated Ha83 protein expression vectors (pET-28a-TC151, pET-28a-TC142, pET-28a-TC128, pET-28a-TC118, pET-28a-TC113, and pET-28a-TC71) were constructed by insertion of the truncated ha83 PCR products into Bam HI and Xho I sites of the pET-28a(+) vector (Novagen, GER) with specific primers (Ha83-F used as forward primer, Ha83C151, Ha83C142, Ha83C128, Ha83C118, Ha83C113, and Ha83C71 were used as reverse primers, respectively) ( ). pET-28a-Ha83 which can encode the entire Ha83 was constructed in a similar way with primers Ha83-F and Ha83-R. And pET-28a-ha81, which contains the entire CDs of ha81 gene, was constructed with primers Ha81-F and Ha81-R as described above and used as the negative control plasmid for ha81 sharing a similar base pair number with ha83 .

    Techniques: Confocal Microscopy, Transfection, Labeling, Staining

    Chitin binding tests on Ha83 and truncated mutants. ( A ) SDS-PAGE investigation of Ha83 and truncated mutants expressed by E. coli BL21(DE3). The protein samples were separated by 15% SDS-PAGE. ( B ) Western blot analyses of Ha83 and truncated mutants expressed by E. coli BL21(DE3). The protein samples were first separated by 10% Tricine-SDS-PAGE and then transferred to a nitrocellulose membrane. A monoclonal anti-His tag antibody (1:4000) (Abcam, GBR) was used as the primary antibodies and the signal was visualized with an Enhanced HRP-DAB Chromogenic Substrate Kit (TIANGEN, CHN). ( C ) Chitin binding tests of Ha83 and truncated mutants incubated with chitin colloid for 16 h at 4 ℃. The bound protein ( B ) and unbound protein in the supernatant ( F ) was first separated by centrifugation then lyophilized and loaded in each lane of a 10% Tricine-SDS-PAGE system. The gels were stained with Coomassie Blue (Sigma). In order to improve the clarity and conciseness, the figures exhibited here were cropped; the full-length gels or blots are presented in Supplementary Figure S2–5 . ( D ) Temporal course of protein samples binding to crab shell chitin. Each chitin binding test was performed in triplicate and average quantity of bound protein was derived from three data measurements for each time point post incubation. Error bars represent the standard deviation. ( E ) Double-reciprocal plot of protein binding to chitin. A standard curve of Ha83 quantity and absorbance figure is zoomed out in the left up corner. The slope and R 2 value of each reciprocal line of protein samples are shown in the table at the bottom of the figure.

    Journal: Scientific Reports

    Article Title: Ha83, a Chitin Binding Domain Encoding Gene, Is Important to Helicoverpa armigera Nucleopolyhedrovirus Budded Virus Production and Occlusion Body Assembling

    doi: 10.1038/srep11088

    Figure Lengend Snippet: Chitin binding tests on Ha83 and truncated mutants. ( A ) SDS-PAGE investigation of Ha83 and truncated mutants expressed by E. coli BL21(DE3). The protein samples were separated by 15% SDS-PAGE. ( B ) Western blot analyses of Ha83 and truncated mutants expressed by E. coli BL21(DE3). The protein samples were first separated by 10% Tricine-SDS-PAGE and then transferred to a nitrocellulose membrane. A monoclonal anti-His tag antibody (1:4000) (Abcam, GBR) was used as the primary antibodies and the signal was visualized with an Enhanced HRP-DAB Chromogenic Substrate Kit (TIANGEN, CHN). ( C ) Chitin binding tests of Ha83 and truncated mutants incubated with chitin colloid for 16 h at 4 ℃. The bound protein ( B ) and unbound protein in the supernatant ( F ) was first separated by centrifugation then lyophilized and loaded in each lane of a 10% Tricine-SDS-PAGE system. The gels were stained with Coomassie Blue (Sigma). In order to improve the clarity and conciseness, the figures exhibited here were cropped; the full-length gels or blots are presented in Supplementary Figure S2–5 . ( D ) Temporal course of protein samples binding to crab shell chitin. Each chitin binding test was performed in triplicate and average quantity of bound protein was derived from three data measurements for each time point post incubation. Error bars represent the standard deviation. ( E ) Double-reciprocal plot of protein binding to chitin. A standard curve of Ha83 quantity and absorbance figure is zoomed out in the left up corner. The slope and R 2 value of each reciprocal line of protein samples are shown in the table at the bottom of the figure.

    Article Snippet: Six truncated Ha83 protein expression vectors (pET-28a-TC151, pET-28a-TC142, pET-28a-TC128, pET-28a-TC118, pET-28a-TC113, and pET-28a-TC71) were constructed by insertion of the truncated ha83 PCR products into Bam HI and Xho I sites of the pET-28a(+) vector (Novagen, GER) with specific primers (Ha83-F used as forward primer, Ha83C151, Ha83C142, Ha83C128, Ha83C118, Ha83C113, and Ha83C71 were used as reverse primers, respectively) ( ). pET-28a-Ha83 which can encode the entire Ha83 was constructed in a similar way with primers Ha83-F and Ha83-R. And pET-28a-ha81, which contains the entire CDs of ha81 gene, was constructed with primers Ha81-F and Ha81-R as described above and used as the negative control plasmid for ha81 sharing a similar base pair number with ha83 .

    Techniques: Binding Assay, SDS Page, Western Blot, Incubation, Centrifugation, Staining, Derivative Assay, Standard Deviation, Protein Binding

    Ha83 deletion analysis in HzAM1 cells. ( A ) Microscopy analysis of viral replication in HzAM1 cells. Fluorescence microscopy shows the progression of viral infection in HzAM1 cells transfected with HaWT ( polyhedrin repaired HaBacHZ8 bacmid), Ha83KO ( ha83 deleted HearNPV mutant), and Ha83Rep (Ha83KO reinserted a ha83 in the polyhedrin locus) from 24 to 72 h p.t. An additional infection fluorescence microscopy shows the infectivity of viral infection in HzAM1 cells infected with HaWT, Ha83KO, and Ha83Rep at 72 h p.i. Light microscopy shows the formation of occlusion bodies in HaWT, Ha83KO, and Ha83Rep infected cells at 96 h p.i. ( B ) Virus growth curves as determined by TCID 50 endpoint dilution assays. For the transfection growth curves, HzAM1 cells were transfected with HaWT, Ha83KO, and Ha83Rep bacmid DNA. The supernatants were then harvested at the indicated time points p. t., and titers were determined using TCID 50 assays. Each data point was determined from the average of three independent transfections, and error bars represent the standard deviations. ( C ) Real-time PCR analysis of viral DNA replication. HzAM1 cells were transfected with each bacmid DNA (HaWT, Ha83KO, or Ha83Rep). At the designated time points, total intracellular DNA was extracted and analyzed by real-time PCR. The y-axis value indicates the number of viral DNA genome copies within each sample. The graph shows the results of three independent replication assays and each replication was tested for another three times, with error bars indicating the standard deviations. Statistically significant differences are indicated by asterisks (* P ⊠ 0.05).

    Journal: Scientific Reports

    Article Title: Ha83, a Chitin Binding Domain Encoding Gene, Is Important to Helicoverpa armigera Nucleopolyhedrovirus Budded Virus Production and Occlusion Body Assembling

    doi: 10.1038/srep11088

    Figure Lengend Snippet: Ha83 deletion analysis in HzAM1 cells. ( A ) Microscopy analysis of viral replication in HzAM1 cells. Fluorescence microscopy shows the progression of viral infection in HzAM1 cells transfected with HaWT ( polyhedrin repaired HaBacHZ8 bacmid), Ha83KO ( ha83 deleted HearNPV mutant), and Ha83Rep (Ha83KO reinserted a ha83 in the polyhedrin locus) from 24 to 72 h p.t. An additional infection fluorescence microscopy shows the infectivity of viral infection in HzAM1 cells infected with HaWT, Ha83KO, and Ha83Rep at 72 h p.i. Light microscopy shows the formation of occlusion bodies in HaWT, Ha83KO, and Ha83Rep infected cells at 96 h p.i. ( B ) Virus growth curves as determined by TCID 50 endpoint dilution assays. For the transfection growth curves, HzAM1 cells were transfected with HaWT, Ha83KO, and Ha83Rep bacmid DNA. The supernatants were then harvested at the indicated time points p. t., and titers were determined using TCID 50 assays. Each data point was determined from the average of three independent transfections, and error bars represent the standard deviations. ( C ) Real-time PCR analysis of viral DNA replication. HzAM1 cells were transfected with each bacmid DNA (HaWT, Ha83KO, or Ha83Rep). At the designated time points, total intracellular DNA was extracted and analyzed by real-time PCR. The y-axis value indicates the number of viral DNA genome copies within each sample. The graph shows the results of three independent replication assays and each replication was tested for another three times, with error bars indicating the standard deviations. Statistically significant differences are indicated by asterisks (* P ⊠ 0.05).

    Article Snippet: Six truncated Ha83 protein expression vectors (pET-28a-TC151, pET-28a-TC142, pET-28a-TC128, pET-28a-TC118, pET-28a-TC113, and pET-28a-TC71) were constructed by insertion of the truncated ha83 PCR products into Bam HI and Xho I sites of the pET-28a(+) vector (Novagen, GER) with specific primers (Ha83-F used as forward primer, Ha83C151, Ha83C142, Ha83C128, Ha83C118, Ha83C113, and Ha83C71 were used as reverse primers, respectively) ( ). pET-28a-Ha83 which can encode the entire Ha83 was constructed in a similar way with primers Ha83-F and Ha83-R. And pET-28a-ha81, which contains the entire CDs of ha81 gene, was constructed with primers Ha81-F and Ha81-R as described above and used as the negative control plasmid for ha81 sharing a similar base pair number with ha83 .

    Techniques: Microscopy, Fluorescence, Infection, Transfection, Mutagenesis, Light Microscopy, Real-time Polymerase Chain Reaction

    Relative expression of viral genes in Ha83KO and HaWT transfected HzAM1 cells. ( A ) Heatmaps of selected gene expression in Ha83KO or HaWT transfected HzAM1 cells at 24, 48, and 72 h p.t. ( B ) Quantification of transcripts from selected viral genes by qPCR. ( a ) polyhedrin , ( b ) p10 , ( c ) cg30 , ( d ) gp41 , ( e ) pif-2 , ( f ) p24 . For each graph, the y-axis indicates the relative amount of RNA estimated in comparison to 1.0 μg of RNA purified from infected HzAM1 cells. Error bars represent the standard deviation. Statistically significant differences between the ha83 knockout and control virus are indicated by asterisks (** P

    Journal: Scientific Reports

    Article Title: Ha83, a Chitin Binding Domain Encoding Gene, Is Important to Helicoverpa armigera Nucleopolyhedrovirus Budded Virus Production and Occlusion Body Assembling

    doi: 10.1038/srep11088

    Figure Lengend Snippet: Relative expression of viral genes in Ha83KO and HaWT transfected HzAM1 cells. ( A ) Heatmaps of selected gene expression in Ha83KO or HaWT transfected HzAM1 cells at 24, 48, and 72 h p.t. ( B ) Quantification of transcripts from selected viral genes by qPCR. ( a ) polyhedrin , ( b ) p10 , ( c ) cg30 , ( d ) gp41 , ( e ) pif-2 , ( f ) p24 . For each graph, the y-axis indicates the relative amount of RNA estimated in comparison to 1.0 μg of RNA purified from infected HzAM1 cells. Error bars represent the standard deviation. Statistically significant differences between the ha83 knockout and control virus are indicated by asterisks (** P

    Article Snippet: Six truncated Ha83 protein expression vectors (pET-28a-TC151, pET-28a-TC142, pET-28a-TC128, pET-28a-TC118, pET-28a-TC113, and pET-28a-TC71) were constructed by insertion of the truncated ha83 PCR products into Bam HI and Xho I sites of the pET-28a(+) vector (Novagen, GER) with specific primers (Ha83-F used as forward primer, Ha83C151, Ha83C142, Ha83C128, Ha83C118, Ha83C113, and Ha83C71 were used as reverse primers, respectively) ( ). pET-28a-Ha83 which can encode the entire Ha83 was constructed in a similar way with primers Ha83-F and Ha83-R. And pET-28a-ha81, which contains the entire CDs of ha81 gene, was constructed with primers Ha81-F and Ha81-R as described above and used as the negative control plasmid for ha81 sharing a similar base pair number with ha83 .

    Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Purification, Infection, Standard Deviation, Knock-Out

    3′RACE and 5′RACE analysis of ha83 in HearNPV infected HzAM1 cells. ( A ) 3′RACE analysis of ha83 in HearNPV infected HzAM1 cells at 48 h p.i. The arrow points to the location of TAA stop codon. The poly A tail addition site is underlined. ( B ) Sequences located at the 5′end of ha83 . The TATA box, ATAAG, TAGG, and CATT promoter motifs are underlined. The rightward arrow indicates the location of the transcription initiation sites. The arrow points to the ATG translation start codon of ha83 .

    Journal: Scientific Reports

    Article Title: Ha83, a Chitin Binding Domain Encoding Gene, Is Important to Helicoverpa armigera Nucleopolyhedrovirus Budded Virus Production and Occlusion Body Assembling

    doi: 10.1038/srep11088

    Figure Lengend Snippet: 3′RACE and 5′RACE analysis of ha83 in HearNPV infected HzAM1 cells. ( A ) 3′RACE analysis of ha83 in HearNPV infected HzAM1 cells at 48 h p.i. The arrow points to the location of TAA stop codon. The poly A tail addition site is underlined. ( B ) Sequences located at the 5′end of ha83 . The TATA box, ATAAG, TAGG, and CATT promoter motifs are underlined. The rightward arrow indicates the location of the transcription initiation sites. The arrow points to the ATG translation start codon of ha83 .

    Article Snippet: Six truncated Ha83 protein expression vectors (pET-28a-TC151, pET-28a-TC142, pET-28a-TC128, pET-28a-TC118, pET-28a-TC113, and pET-28a-TC71) were constructed by insertion of the truncated ha83 PCR products into Bam HI and Xho I sites of the pET-28a(+) vector (Novagen, GER) with specific primers (Ha83-F used as forward primer, Ha83C151, Ha83C142, Ha83C128, Ha83C118, Ha83C113, and Ha83C71 were used as reverse primers, respectively) ( ). pET-28a-Ha83 which can encode the entire Ha83 was constructed in a similar way with primers Ha83-F and Ha83-R. And pET-28a-ha81, which contains the entire CDs of ha81 gene, was constructed with primers Ha81-F and Ha81-R as described above and used as the negative control plasmid for ha81 sharing a similar base pair number with ha83 .

    Techniques: Infection

    SDS-PAGE and Western blot analysis of the expression of recombinant Ifu-Chit2 in E. coli . Lane M: pre-stained protein marker; Lane 1: total protein of cells containing empty vector pET-28a with IPTG induction; Lane 2: total protein of cells containing expression vector pET- 28a-Ifu-chit2 without IPTG induction; Lanes 3–8: total protein of cells containing expression vector pET- 28a-Ifu-chit2 induced by IPTG at 28 °C for 1, 2, 3, 4, 5 and 6 h, respectively; Lane 9: purified recombinant Ifu-Chit2 (50 kDa). Lane 10: total protein of cells containing empty vector pET-28a with IPTG induction; Lane 11: total protein of cells containing expression vector pET- 28a-Ifu-chit2 without IPTG induction; Lane 12: total protein of cells containing expression vector pET- 28a-Ifu-chit2 induced by IPTG at 28 °C for 4 h; Lane13: Purified recombinant Ifu-Chit2.

    Journal: Genetics and Molecular Biology

    Article Title: Cloning and expression analysis of the chitinase gene Ifu-chit2 from Isaria fumosorosea

    doi: 10.1590/S1415-475738320150003

    Figure Lengend Snippet: SDS-PAGE and Western blot analysis of the expression of recombinant Ifu-Chit2 in E. coli . Lane M: pre-stained protein marker; Lane 1: total protein of cells containing empty vector pET-28a with IPTG induction; Lane 2: total protein of cells containing expression vector pET- 28a-Ifu-chit2 without IPTG induction; Lanes 3–8: total protein of cells containing expression vector pET- 28a-Ifu-chit2 induced by IPTG at 28 °C for 1, 2, 3, 4, 5 and 6 h, respectively; Lane 9: purified recombinant Ifu-Chit2 (50 kDa). Lane 10: total protein of cells containing empty vector pET-28a with IPTG induction; Lane 11: total protein of cells containing expression vector pET- 28a-Ifu-chit2 without IPTG induction; Lane 12: total protein of cells containing expression vector pET- 28a-Ifu-chit2 induced by IPTG at 28 °C for 4 h; Lane13: Purified recombinant Ifu-Chit2.

    Article Snippet: The PCR product was cloned into the pET-28a (+) vector (Novagen), resulting in recombinant expression vector pET-28a-Ifu-chit2 .

    Techniques: SDS Page, Western Blot, Expressing, Recombinant, Staining, Marker, Plasmid Preparation, Positron Emission Tomography, Purification

    Agarose gel showing confirmation of insert in pET 28a. lane 1 is 100 bp DNA ladder, lane 2 is uncut pET 28a-Ndk construct and lane 3 is restriction digestion of pET 28a-Ndk gene.

    Journal: Enzyme Research

    Article Title: Cloning, Expression, and Purification of Nucleoside Diphosphate Kinase from Acinetobacter baumannii

    doi: 10.1155/2013/597028

    Figure Lengend Snippet: Agarose gel showing confirmation of insert in pET 28a. lane 1 is 100 bp DNA ladder, lane 2 is uncut pET 28a-Ndk construct and lane 3 is restriction digestion of pET 28a-Ndk gene.

    Article Snippet: Plasmid pET-28a (Novagen, Wisconsin, USA) was used as an expression vector.

    Techniques: Agarose Gel Electrophoresis, Positron Emission Tomography, Construct

    Microscopy images illustrating CatIB/FIB formation in E. coli . a – f Phase-contrast and fluorescence images of E. coli cells expressing a TDoT-L-YFP, b TDoT-YFP, c 3HAMP-L-YFP, d TDoT-L-mCherry, e TDoT-mCherry, and f 3HAMP-mCherry. g – k phase-contrast images of TDoT fusion (left) and 3HAMP fusion (right) expressing E. coli cells containing g R ADH, h Lb ADH, i Pf BAL, j Pp BFD, k Ec LDC (here, the coiled-coil domain is fused C-terminally), and l E. coli BL21(DE3) with empty pET-28a vector. All strains were grown under standard growth conditions as described in “ Methods ” section

    Journal: Microbial Cell Factories

    Article Title: Tailoring the properties of (catalytically)-active inclusion bodies

    doi: 10.1186/s12934-019-1081-5

    Figure Lengend Snippet: Microscopy images illustrating CatIB/FIB formation in E. coli . a – f Phase-contrast and fluorescence images of E. coli cells expressing a TDoT-L-YFP, b TDoT-YFP, c 3HAMP-L-YFP, d TDoT-L-mCherry, e TDoT-mCherry, and f 3HAMP-mCherry. g – k phase-contrast images of TDoT fusion (left) and 3HAMP fusion (right) expressing E. coli cells containing g R ADH, h Lb ADH, i Pf BAL, j Pp BFD, k Ec LDC (here, the coiled-coil domain is fused C-terminally), and l E. coli BL21(DE3) with empty pET-28a vector. All strains were grown under standard growth conditions as described in “ Methods ” section

    Article Snippet: If not stated otherwise, all gene fusions consisted of gene fragments coding for a coiled-coil domain (here TDoT or 3HAMP), a linker polypeptide, consisting of a protease Factor Xa cleavage site and a triple (GGGS)3 and the respective target proteins/enzymes cloned into a pET-28a vector (Novagen, Merck KGaA, Frankfurt, Germany).

    Techniques: Microscopy, Fluorescence, Expressing, Positron Emission Tomography, Plasmid Preparation

    Analysis of rMsEno expression and purification using SDS-PAGE followed by Coomassie blue staining. M: PageRuler™ Prestained Protein Ladder (SM0671, Fermentas). Lane 1: Total cellular proteins of E. coli BL21 (DE3) cells transformed by pET-28a (+). Lane 2: Total cellular proteins of E. coli BL21 (DE3) cells transformed by pET-Eno. Lane 3: Supernatant of lysate of E. coli BL21 (DE3) cells transformed by pET-Eno. Lane 4: Sediment of lysate of E. coli BL21 (DE3) cells transformed by pET-Eno. Lane 5: Purified recombinant protein.

    Journal: BMC Veterinary Research

    Article Title: Mycoplasma synoviae enolase is a plasminogen/fibronectin binding protein

    doi: 10.1186/s12917-014-0223-6

    Figure Lengend Snippet: Analysis of rMsEno expression and purification using SDS-PAGE followed by Coomassie blue staining. M: PageRuler™ Prestained Protein Ladder (SM0671, Fermentas). Lane 1: Total cellular proteins of E. coli BL21 (DE3) cells transformed by pET-28a (+). Lane 2: Total cellular proteins of E. coli BL21 (DE3) cells transformed by pET-Eno. Lane 3: Supernatant of lysate of E. coli BL21 (DE3) cells transformed by pET-Eno. Lane 4: Sediment of lysate of E. coli BL21 (DE3) cells transformed by pET-Eno. Lane 5: Purified recombinant protein.

    Article Snippet: The pET-28a (+) expression vector was obtained from Novagen (Madison, WI, USA).

    Techniques: Expressing, Purification, SDS Page, Staining, Transformation Assay, Positron Emission Tomography, Recombinant