promastigote transformation  (Millipore)


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  • 97
    Name:
    transformation
    Description:
    BL21 T1R are competent E coli that are suitable for high level expression production of heterologous proteins regulated by various expression vector systems The cells have transformation efficiency of 3x106 cfu mug when transformed with non saturating amounts of pUC19 plasmid DNA
    Catalog Number:
    b2685
    Price:
    None
    Applications:
    Suitable for expression and production of proteins directed by a range of expression systems with promoters such as lac, trc, tac, lambdaPL and araD
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    Structured Review

    Millipore promastigote transformation
    BL21 T1R are competent E coli that are suitable for high level expression production of heterologous proteins regulated by various expression vector systems The cells have transformation efficiency of 3x106 cfu mug when transformed with non saturating amounts of pUC19 plasmid DNA
    https://www.bioz.com/result/promastigote transformation/product/Millipore
    Average 97 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    promastigote transformation - by Bioz Stars, 2020-07
    97/100 stars

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    Related Articles

    Dominant Negative Mutation:

    Article Title: AKT regulation of mesothelial-to-mesenchymal transition in peritoneal dialysis is modulated by smurf2 and deubiquitinating enzyme USP4
    Article Snippet: .. To examine the role of Akt in MMT transformation, Smad7 and Smurf2 expression of HPMCs, LY294002 (10–20 μg/ml, Calbiochem) was used to pretreat the cells for 30 min or they were pre-transfected with a dominant negative Akt mutant (K179M) plasmid (Akt-DN, Addgene) for 24 hrs and then the cells were exposed to TGF-β1 (5 ng/ml) for 24–48 hrs. .. To investigate the effect of Smurf2 and USP4 on MMT transformation of HPMCs, HPMCs were transfected with Smurf2 siRNA (Ambion), USP4 siRNA (Santa Cruz Biotech), respectively, with or without pWZL Neo Myr Flag AKT1 (Flag Akt plasmid) which is part of a kinase library, and has constitutively active expression (Biovector – Addgene, Plasmid 20422) for 24 hrs.

    Electroporation:

    Article Title: New capabilities for Mycosphaerella graminicola research
    Article Snippet: .. Agrobacterium tumefaciens strain EHA105 was transformed with the binary vectors for M. graminicola transformation by electroporation following standard procedures, and subsequent strains were maintained in LB medium supplemented with 100 µg/mL spectinomycin (Sigma, Gillingham, Dorset, UK) and 100 µg/mL rifampicin (Sigma). .. Transformed A. tumefaciens strains were grown overnight in LB medium amended with 100 µg/mL spectinomycin and 100 µg/mL rifampicin at 28 °C, 250 r.p.m.

    Transfection:

    Article Title: Regulation of Human Papillomavirus Type 16 E7 Activity through Direct Protein Interaction with the E2 Transcriptional Activator
    Article Snippet: .. Total RNA was isolated from CaSKi cells (transfected as above) and BRK cells (transfected as in the transformation assay) 24 h after transfection with TRI Reagent (Sigma) according to the manufacturer's instructions. .. A total of 1 μg of RNA was subjected to reverse transcription (RT) with the RETROscript system (Ambion).

    Mutagenesis:

    Article Title: AKT regulation of mesothelial-to-mesenchymal transition in peritoneal dialysis is modulated by smurf2 and deubiquitinating enzyme USP4
    Article Snippet: .. To examine the role of Akt in MMT transformation, Smad7 and Smurf2 expression of HPMCs, LY294002 (10–20 μg/ml, Calbiochem) was used to pretreat the cells for 30 min or they were pre-transfected with a dominant negative Akt mutant (K179M) plasmid (Akt-DN, Addgene) for 24 hrs and then the cells were exposed to TGF-β1 (5 ng/ml) for 24–48 hrs. .. To investigate the effect of Smurf2 and USP4 on MMT transformation of HPMCs, HPMCs were transfected with Smurf2 siRNA (Ambion), USP4 siRNA (Santa Cruz Biotech), respectively, with or without pWZL Neo Myr Flag AKT1 (Flag Akt plasmid) which is part of a kinase library, and has constitutively active expression (Biovector – Addgene, Plasmid 20422) for 24 hrs.

    Isolation:

    Article Title: Regulation of Human Papillomavirus Type 16 E7 Activity through Direct Protein Interaction with the E2 Transcriptional Activator
    Article Snippet: .. Total RNA was isolated from CaSKi cells (transfected as above) and BRK cells (transfected as in the transformation assay) 24 h after transfection with TRI Reagent (Sigma) according to the manufacturer's instructions. .. A total of 1 μg of RNA was subjected to reverse transcription (RT) with the RETROscript system (Ambion).

    Activation Assay:

    Article Title: Contractile Forces Contribute to Increased Glycosylphosphatidylinositol-anchored Receptor CD24-facilitated Cancer Cell Invasion
    Article Snippet: .. Furthermore, to analyze the CD24-mediating signaling, we inhibited or modulated cell invasion by addition of 30 μ m Src tyrosine kinase inhibitor (catalog no. 567805, Calbiochem), 30 μ m STAT3 inhibitor peptide (catalog no. 573096, Calbiochem), which blocks the STAT3 activation and suppresses constitutive STAT3-dependent Src transformation, or 100 μ m tyrosine kinase inhibitor herbimycin A (catalog no. 375670, Calbiochem). .. To inhibit enzymatic degradation of the three-dimensional collagen matrices, we added a protease inhibitor (PI) mixture, which also blocks the matrix metalloproteinases, including MT1-MMP, before the start of the invasion assay.

    Cell Transformation Assay:

    Article Title: A natural small molecule, catechol, induces c-Myc degradation by directly targeting ERK2 in lung cancer
    Article Snippet: .. Anchorage-independent cell transformation assay Cells (8 × 103 /well) suspended in 10% Basal Medium Eagle (Sigma Aldrich, St. Louis, MO) were added to 0.3% agar with 0, 10, 20 or 40 μM catechol in a top layer over a base layer of 0.5% agar with 0, 10, 20, or 40 μM catechol. .. The cultures were maintained at 37°C in a 5% CO2 incubator for 1–2 weeks and then colonies were counted under a microscope using the Image-Pro Plus software (v.6.1) program (Media Cybernetics).

    Expressing:

    Article Title: AKT regulation of mesothelial-to-mesenchymal transition in peritoneal dialysis is modulated by smurf2 and deubiquitinating enzyme USP4
    Article Snippet: .. To examine the role of Akt in MMT transformation, Smad7 and Smurf2 expression of HPMCs, LY294002 (10–20 μg/ml, Calbiochem) was used to pretreat the cells for 30 min or they were pre-transfected with a dominant negative Akt mutant (K179M) plasmid (Akt-DN, Addgene) for 24 hrs and then the cells were exposed to TGF-β1 (5 ng/ml) for 24–48 hrs. .. To investigate the effect of Smurf2 and USP4 on MMT transformation of HPMCs, HPMCs were transfected with Smurf2 siRNA (Ambion), USP4 siRNA (Santa Cruz Biotech), respectively, with or without pWZL Neo Myr Flag AKT1 (Flag Akt plasmid) which is part of a kinase library, and has constitutively active expression (Biovector – Addgene, Plasmid 20422) for 24 hrs.

    CRISPR:

    Article Title: Selection of chromosomal DNA libraries using a multiplex CRISPR system
    Article Snippet: .. CRISPR-Cas9 screening protocol The Cas9 transformation mix consisted of 90 µl yeast competent cell mix (OD600 = 1.0), 10.0 µl × 10 mg/ml ssDNA (D9156; Sigma, St. Louis, MO), 1.0 µg pCAS plasmid, 5.0 µg of linear repair DNA and 900 µl Polyethyleneglycol2000 (295906; Sigma), 0.1 M Lithium acetate (517992; Sigma) 0.05 M Tris–HCl (155568; Invitrogen) EDTA (10618973; Fisher Scientific). .. To measure Cas9 independent integration, the linear DNA was co-transformed with a plasmid lacking the Cas9 protein and sgRNA (pOR1.1) ( ).

    Transformation Assay:

    Article Title: Selection of chromosomal DNA libraries using a multiplex CRISPR system
    Article Snippet: .. CRISPR-Cas9 screening protocol The Cas9 transformation mix consisted of 90 µl yeast competent cell mix (OD600 = 1.0), 10.0 µl × 10 mg/ml ssDNA (D9156; Sigma, St. Louis, MO), 1.0 µg pCAS plasmid, 5.0 µg of linear repair DNA and 900 µl Polyethyleneglycol2000 (295906; Sigma), 0.1 M Lithium acetate (517992; Sigma) 0.05 M Tris–HCl (155568; Invitrogen) EDTA (10618973; Fisher Scientific). .. To measure Cas9 independent integration, the linear DNA was co-transformed with a plasmid lacking the Cas9 protein and sgRNA (pOR1.1) ( ).

    Article Title: Evolving thermostability in mutant libraries of ligninolytic oxidoreductases expressed in yeast
    Article Snippet: .. ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), bovine haemoglobine, Taq polymerase and the S. cerevisiae transformation kit were purchased from Sigma-Aldrich (Madrid, Spain). .. The E. coli XL2-blue competent cells and the Genemorph Random mutagenesis kit were from Stratagene (La Jolla, CA, USA).

    Article Title: New capabilities for Mycosphaerella graminicola research
    Article Snippet: .. Agrobacterium tumefaciens strain EHA105 was transformed with the binary vectors for M. graminicola transformation by electroporation following standard procedures, and subsequent strains were maintained in LB medium supplemented with 100 µg/mL spectinomycin (Sigma, Gillingham, Dorset, UK) and 100 µg/mL rifampicin (Sigma). .. Transformed A. tumefaciens strains were grown overnight in LB medium amended with 100 µg/mL spectinomycin and 100 µg/mL rifampicin at 28 °C, 250 r.p.m.

    Article Title: AKT regulation of mesothelial-to-mesenchymal transition in peritoneal dialysis is modulated by smurf2 and deubiquitinating enzyme USP4
    Article Snippet: .. To examine the role of Akt in MMT transformation, Smad7 and Smurf2 expression of HPMCs, LY294002 (10–20 μg/ml, Calbiochem) was used to pretreat the cells for 30 min or they were pre-transfected with a dominant negative Akt mutant (K179M) plasmid (Akt-DN, Addgene) for 24 hrs and then the cells were exposed to TGF-β1 (5 ng/ml) for 24–48 hrs. .. To investigate the effect of Smurf2 and USP4 on MMT transformation of HPMCs, HPMCs were transfected with Smurf2 siRNA (Ambion), USP4 siRNA (Santa Cruz Biotech), respectively, with or without pWZL Neo Myr Flag AKT1 (Flag Akt plasmid) which is part of a kinase library, and has constitutively active expression (Biovector – Addgene, Plasmid 20422) for 24 hrs.

    Article Title: Contractile Forces Contribute to Increased Glycosylphosphatidylinositol-anchored Receptor CD24-facilitated Cancer Cell Invasion
    Article Snippet: .. Furthermore, to analyze the CD24-mediating signaling, we inhibited or modulated cell invasion by addition of 30 μ m Src tyrosine kinase inhibitor (catalog no. 567805, Calbiochem), 30 μ m STAT3 inhibitor peptide (catalog no. 573096, Calbiochem), which blocks the STAT3 activation and suppresses constitutive STAT3-dependent Src transformation, or 100 μ m tyrosine kinase inhibitor herbimycin A (catalog no. 375670, Calbiochem). .. To inhibit enzymatic degradation of the three-dimensional collagen matrices, we added a protease inhibitor (PI) mixture, which also blocks the matrix metalloproteinases, including MT1-MMP, before the start of the invasion assay.

    Article Title: Activation of σ20-dependent recombination and horizontal gene transfer in Mycoplasma genitalium
    Article Snippet: .. Plasmids for M. genitalium transformation were obtained using the GenElute HP Midiprep kit (Sigma). .. Mutant construction, transformation and screening A detailed explanation of the steps and methodology, including primers and plasmids, used to construct all the mutants created in this study is supplied as .

    Article Title: Regulation of Human Papillomavirus Type 16 E7 Activity through Direct Protein Interaction with the E2 Transcriptional Activator
    Article Snippet: .. Total RNA was isolated from CaSKi cells (transfected as above) and BRK cells (transfected as in the transformation assay) 24 h after transfection with TRI Reagent (Sigma) according to the manufacturer's instructions. .. A total of 1 μg of RNA was subjected to reverse transcription (RT) with the RETROscript system (Ambion).

    Plasmid Preparation:

    Article Title: Selection of chromosomal DNA libraries using a multiplex CRISPR system
    Article Snippet: .. CRISPR-Cas9 screening protocol The Cas9 transformation mix consisted of 90 µl yeast competent cell mix (OD600 = 1.0), 10.0 µl × 10 mg/ml ssDNA (D9156; Sigma, St. Louis, MO), 1.0 µg pCAS plasmid, 5.0 µg of linear repair DNA and 900 µl Polyethyleneglycol2000 (295906; Sigma), 0.1 M Lithium acetate (517992; Sigma) 0.05 M Tris–HCl (155568; Invitrogen) EDTA (10618973; Fisher Scientific). .. To measure Cas9 independent integration, the linear DNA was co-transformed with a plasmid lacking the Cas9 protein and sgRNA (pOR1.1) ( ).

    Article Title: AKT regulation of mesothelial-to-mesenchymal transition in peritoneal dialysis is modulated by smurf2 and deubiquitinating enzyme USP4
    Article Snippet: .. To examine the role of Akt in MMT transformation, Smad7 and Smurf2 expression of HPMCs, LY294002 (10–20 μg/ml, Calbiochem) was used to pretreat the cells for 30 min or they were pre-transfected with a dominant negative Akt mutant (K179M) plasmid (Akt-DN, Addgene) for 24 hrs and then the cells were exposed to TGF-β1 (5 ng/ml) for 24–48 hrs. .. To investigate the effect of Smurf2 and USP4 on MMT transformation of HPMCs, HPMCs were transfected with Smurf2 siRNA (Ambion), USP4 siRNA (Santa Cruz Biotech), respectively, with or without pWZL Neo Myr Flag AKT1 (Flag Akt plasmid) which is part of a kinase library, and has constitutively active expression (Biovector – Addgene, Plasmid 20422) for 24 hrs.

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  • 96
    Millipore monastrol
    Effect of Eg5 inhibition on the porcine oocyte maturation. A Representative images of control and Eg5-inhibited oocytes cultured in vitro for 44 h. Cumulus cell expansion of COCs and polar body extrusion of DOs were imaged by confocal microscope. Scale bar, 150 μm ( a , b ); 80 μm ( c , d ); 20 μm ( e , f ). B The rates of polar body extrusion were recorded in control and different concentrations (10, 50 and 100 μM) of <t>monastrol-treated</t> oocytes. C The percentage of control and Eg5-inhibited oocytes in GV, M I or M II stage after in vitro maturation. D Oocytes that underwent GVBD were recorded in control and monastrol-treated groups. Scale bar, 5 μm. Data of B – D were presented as mean percentage (mean ± SEM) of at least three independent experiments. ** p
    Monastrol, supplied by Millipore, used in various techniques. Bioz Stars score: 96/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monastrol/product/Millipore
    Average 96 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    monastrol - by Bioz Stars, 2020-07
    96/100 stars
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    90
    Millipore l infantum promastigotes
    Immunochemical detection of Ld-mao1 and Ld-ppi1 in L. donovani and L. <t>infantum</t> <t>promastigotes.</t> Validation of Ld-mao1 and Ld-ppi1 proteins as genuine molecules produced by L. donovani and L. infantum was performed by Western blotting using promastigote
    L Infantum Promastigotes, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/l infantum promastigotes/product/Millipore
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    l infantum promastigotes - by Bioz Stars, 2020-07
    90/100 stars
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    89
    Millipore l donovani promastigotes
    Determination of expression level of LdTryS in exponential vs. stationary phase of L. <t>donovani.</t> (A) Semiquantitative RT-PCR analysis of LdTryS transcript in exponential and stationary phase <t>promastigotes.</t> Ethidium bromide-stained PCR products were photographed and the image was analyzed densitometrically. α-tubulin was used as control to show uniform expression of a housekeeping gene in both stages of promastigotes. (B) Bar graph represents quantitative real time PCR analysis of LdTryS expression level in exponential vs. stationary phase promastigotes. Data are normalized by the target/reference ratio of the calibrator. (C) Western blot of 30 µg total Leishmania lysate proteins from exponential phase (EP) and stationary phase (SP), and image was analyzed densitometrically. Data was normalized and β-actin was used as control. (D) Western blot of increasing quantities of recombinant LdTryS used as standard and analyzed densitometrically to compare the expression level of TryS in Leishmania lysate. The experiments were repeated thrice and data represents the mean ± SD.
    L Donovani Promastigotes, supplied by Millipore, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/l donovani promastigotes/product/Millipore
    Average 89 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    l donovani promastigotes - by Bioz Stars, 2020-07
    89/100 stars
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    Image Search Results


    Effect of Eg5 inhibition on the porcine oocyte maturation. A Representative images of control and Eg5-inhibited oocytes cultured in vitro for 44 h. Cumulus cell expansion of COCs and polar body extrusion of DOs were imaged by confocal microscope. Scale bar, 150 μm ( a , b ); 80 μm ( c , d ); 20 μm ( e , f ). B The rates of polar body extrusion were recorded in control and different concentrations (10, 50 and 100 μM) of monastrol-treated oocytes. C The percentage of control and Eg5-inhibited oocytes in GV, M I or M II stage after in vitro maturation. D Oocytes that underwent GVBD were recorded in control and monastrol-treated groups. Scale bar, 5 μm. Data of B – D were presented as mean percentage (mean ± SEM) of at least three independent experiments. ** p

    Journal: Cell Division

    Article Title: Eg5 orchestrates porcine oocyte maturational progression by maintaining meiotic organelle arrangement

    doi: 10.1186/s13008-018-0037-1

    Figure Lengend Snippet: Effect of Eg5 inhibition on the porcine oocyte maturation. A Representative images of control and Eg5-inhibited oocytes cultured in vitro for 44 h. Cumulus cell expansion of COCs and polar body extrusion of DOs were imaged by confocal microscope. Scale bar, 150 μm ( a , b ); 80 μm ( c , d ); 20 μm ( e , f ). B The rates of polar body extrusion were recorded in control and different concentrations (10, 50 and 100 μM) of monastrol-treated oocytes. C The percentage of control and Eg5-inhibited oocytes in GV, M I or M II stage after in vitro maturation. D Oocytes that underwent GVBD were recorded in control and monastrol-treated groups. Scale bar, 5 μm. Data of B – D were presented as mean percentage (mean ± SEM) of at least three independent experiments. ** p

    Article Snippet: Monastrol (Calbiochem, San Diego, CA, USA) was dissolved in DMSO and diluted into a final concentration of 10, 50 and 100 μM, respectively, with maturation medium, with the final concentration of the solvent not more than 0.1% of the culture medium.

    Techniques: Inhibition, Cell Culture, In Vitro, Microscopy

    AURKC splice variants differ in catalytic activity. Fully grown oocytes arrested at prophase of MI from Aurkc −/− mice were injected with the indicated Gfp -tagged cRNA and matured to Met I. ( A – C ) Oocytes were fixed and immunocytochemistry was used to detect pINCENP (red in merge). (A) Representative confocal z-projections. DNA was detected by DAPI staining (blue). Detection of GFP is green in the merge. Scale bars represent 10 µm. (B) Quantification of experiments in (A) after normalization with the intensity in the Gfp control group. (C) Quantification of chromosome alignment from experiments in (A) as described in Lane et al and our Materials and Methods section. Each experiment was performed three times using two mice each time. A permutation version of the binomial proportions test was used to analyze differences between groups as described in Materials and Methods section. * P = 0.0057. Variants 2 and 3 were not significantly different from the Gfp control ( P = 0.7233, and 0.8697, respectively). ( D – F ) After maturation to Met I, oocytes were treated with monastrol for 2 h followed by a 3 h recovery. Prior to fixation, oocytes were incubated in ice-cold medium. After fixation, stable K-MT attachments were detected by immunocytochemistry to visualize spindle fibers (α-tubulin; green) and kinetochores (CREST; red). (D and E) Each data point represents the number of improper K-MT attachments quantified in a single oocyte image. (F) Representative confocal z-projections of types of K-MT attachments. Full images are shown in Supplementary data, Fig. S2 . Normal attachments are classified as a chromosome containing two pairs of sister kinetochores attached to opposite poles. Syntelic attachments occur when both pairs of sister kinetochores are attached to the same pole. Merotelic attachments occur when one pair of sister kinetochores is attached to both poles. The scale bar is 5 µm. The experiments were performed twice with a total of four mice (D) or three times with a total of five mice (E). One-way ANOVA was used to analyze the data and error bars represent the mean (±SEM); * P = 0.0143 and 0.0329, respectively, ** P = 0.007, *** P = 0.0002, **** P

    Journal: Molecular Human Reproduction

    Article Title: Expression and characterization of three Aurora kinase C splice variants found in human oocytes

    doi: 10.1093/molehr/gav026

    Figure Lengend Snippet: AURKC splice variants differ in catalytic activity. Fully grown oocytes arrested at prophase of MI from Aurkc −/− mice were injected with the indicated Gfp -tagged cRNA and matured to Met I. ( A – C ) Oocytes were fixed and immunocytochemistry was used to detect pINCENP (red in merge). (A) Representative confocal z-projections. DNA was detected by DAPI staining (blue). Detection of GFP is green in the merge. Scale bars represent 10 µm. (B) Quantification of experiments in (A) after normalization with the intensity in the Gfp control group. (C) Quantification of chromosome alignment from experiments in (A) as described in Lane et al and our Materials and Methods section. Each experiment was performed three times using two mice each time. A permutation version of the binomial proportions test was used to analyze differences between groups as described in Materials and Methods section. * P = 0.0057. Variants 2 and 3 were not significantly different from the Gfp control ( P = 0.7233, and 0.8697, respectively). ( D – F ) After maturation to Met I, oocytes were treated with monastrol for 2 h followed by a 3 h recovery. Prior to fixation, oocytes were incubated in ice-cold medium. After fixation, stable K-MT attachments were detected by immunocytochemistry to visualize spindle fibers (α-tubulin; green) and kinetochores (CREST; red). (D and E) Each data point represents the number of improper K-MT attachments quantified in a single oocyte image. (F) Representative confocal z-projections of types of K-MT attachments. Full images are shown in Supplementary data, Fig. S2 . Normal attachments are classified as a chromosome containing two pairs of sister kinetochores attached to opposite poles. Syntelic attachments occur when both pairs of sister kinetochores are attached to the same pole. Merotelic attachments occur when one pair of sister kinetochores is attached to both poles. The scale bar is 5 µm. The experiments were performed twice with a total of four mice (D) or three times with a total of five mice (E). One-way ANOVA was used to analyze the data and error bars represent the mean (±SEM); * P = 0.0143 and 0.0329, respectively, ** P = 0.007, *** P = 0.0002, **** P

    Article Snippet: After 2 h oocytes were washed out of the monastrol-containing media and allowed to recover in CZB containing 5 µM MG132 (Calbiochem, #47 4791) to prevent anaphase onset.

    Techniques: Activity Assay, Mouse Assay, Injection, Immunocytochemistry, Staining, Incubation

    Immunochemical detection of Ld-mao1 and Ld-ppi1 in L. donovani and L. infantum promastigotes. Validation of Ld-mao1 and Ld-ppi1 proteins as genuine molecules produced by L. donovani and L. infantum was performed by Western blotting using promastigote

    Journal: Journal of Clinical Microbiology

    Article Title: Development of a Multiplexed Assay for Detection of Leishmania donovani and Leishmania infantum Protein Biomarkers in Urine Samples of Patients with Visceral Leishmaniasis

    doi: 10.1128/JCM.02076-18

    Figure Lengend Snippet: Immunochemical detection of Ld-mao1 and Ld-ppi1 in L. donovani and L. infantum promastigotes. Validation of Ld-mao1 and Ld-ppi1 proteins as genuine molecules produced by L. donovani and L. infantum was performed by Western blotting using promastigote

    Article Snippet: Purified recombinant proteins (100 ng) and whole-lysate extract from L. donovani and L. infantum promastigotes were fractionated by SDS-PAGE (4% to 20% gradient gel) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Medford, MA).

    Techniques: Produced, Western Blot

    Determination of expression level of LdTryS in exponential vs. stationary phase of L. donovani. (A) Semiquantitative RT-PCR analysis of LdTryS transcript in exponential and stationary phase promastigotes. Ethidium bromide-stained PCR products were photographed and the image was analyzed densitometrically. α-tubulin was used as control to show uniform expression of a housekeeping gene in both stages of promastigotes. (B) Bar graph represents quantitative real time PCR analysis of LdTryS expression level in exponential vs. stationary phase promastigotes. Data are normalized by the target/reference ratio of the calibrator. (C) Western blot of 30 µg total Leishmania lysate proteins from exponential phase (EP) and stationary phase (SP), and image was analyzed densitometrically. Data was normalized and β-actin was used as control. (D) Western blot of increasing quantities of recombinant LdTryS used as standard and analyzed densitometrically to compare the expression level of TryS in Leishmania lysate. The experiments were repeated thrice and data represents the mean ± SD.

    Journal: PLoS ONE

    Article Title: Stage-Dependent Expression and Up-Regulation of Trypanothione Synthetase in Amphotericin B Resistant Leishmania donovani

    doi: 10.1371/journal.pone.0097600

    Figure Lengend Snippet: Determination of expression level of LdTryS in exponential vs. stationary phase of L. donovani. (A) Semiquantitative RT-PCR analysis of LdTryS transcript in exponential and stationary phase promastigotes. Ethidium bromide-stained PCR products were photographed and the image was analyzed densitometrically. α-tubulin was used as control to show uniform expression of a housekeeping gene in both stages of promastigotes. (B) Bar graph represents quantitative real time PCR analysis of LdTryS expression level in exponential vs. stationary phase promastigotes. Data are normalized by the target/reference ratio of the calibrator. (C) Western blot of 30 µg total Leishmania lysate proteins from exponential phase (EP) and stationary phase (SP), and image was analyzed densitometrically. Data was normalized and β-actin was used as control. (D) Western blot of increasing quantities of recombinant LdTryS used as standard and analyzed densitometrically to compare the expression level of TryS in Leishmania lysate. The experiments were repeated thrice and data represents the mean ± SD.

    Article Snippet: Digitonin Fractionation of L. donovani Promastigotes The differential membrane permeabilization of L. donovani promastigotes was done using digitonin (Calbiochem), as described previously , .

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot, Recombinant

    Up regulation of LdTryS in L. donovani promastigotes in response to H 2 O 2 treatment. (A) TryS expression in L. donovani parasites in the presence of H 2 O 2 (10–200 µM) was analysed by western blot. H 2 O 2 treated parasites showed increased expression level of LdTryS, whereas the β-actin expression level did not change significantly. The experiments were repeated twice in duplicates and quantitation was done by densitometric analysis using Quantity One (Bio-Rad). Band intensity is presented as fold increase/decrease of LdTryS expression. (B) LdTryS expression level was analyzed by semiquantitative RT-PCR, and PCR product stained with ethidium bromide and photographed. PCR of α-tubulin was used as housekeeping control that showed uniform expression pattern irrespective of H 2 O 2 concentration.

    Journal: PLoS ONE

    Article Title: Stage-Dependent Expression and Up-Regulation of Trypanothione Synthetase in Amphotericin B Resistant Leishmania donovani

    doi: 10.1371/journal.pone.0097600

    Figure Lengend Snippet: Up regulation of LdTryS in L. donovani promastigotes in response to H 2 O 2 treatment. (A) TryS expression in L. donovani parasites in the presence of H 2 O 2 (10–200 µM) was analysed by western blot. H 2 O 2 treated parasites showed increased expression level of LdTryS, whereas the β-actin expression level did not change significantly. The experiments were repeated twice in duplicates and quantitation was done by densitometric analysis using Quantity One (Bio-Rad). Band intensity is presented as fold increase/decrease of LdTryS expression. (B) LdTryS expression level was analyzed by semiquantitative RT-PCR, and PCR product stained with ethidium bromide and photographed. PCR of α-tubulin was used as housekeeping control that showed uniform expression pattern irrespective of H 2 O 2 concentration.

    Article Snippet: Digitonin Fractionation of L. donovani Promastigotes The differential membrane permeabilization of L. donovani promastigotes was done using digitonin (Calbiochem), as described previously , .

    Techniques: Expressing, Western Blot, Quantitation Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Staining, Concentration Assay

    Determination of expression level of LdTryS in sensitive (S) vs. drug resistant (R) strains of L. donovani. (A) Semiquantitative RT-PCR analysis of LdTryS transcript in Amp B sensitive vs. resistant isolates. Ethidium bromide-stained PCR products were photographed and the image was analyzed densitometrically. α-tubulin was used as control to show uniform expression of a housekeeping gene in both Amp B sensitive and resistant promastigotes. (B) Bar graph repr esents quantitative real time PCR analysis of LdTryS expression level in Amp B sensitive vs. resistant isolates. Data are normalized by the target/reference ratio of the calibrator. (C) The total Leishmania lysates (30 µg) were electrophoresed on 10% SDS-PAGE gel and stained with coomassie brilliant blue. Lane 1 represents, protein marker; lane 2 represents, sensitive strain (S); lanes 3, and 4 represent, resistant isolates (R1, R2). (D) Shows western blot of same coomassie gel using anti-LdTryS (1∶3000). The image was analyzed by densitometrically. Data was normalized and β-actin was used as control. The experiments were repeated twice and graphs represent the mean ± SD.

    Journal: PLoS ONE

    Article Title: Stage-Dependent Expression and Up-Regulation of Trypanothione Synthetase in Amphotericin B Resistant Leishmania donovani

    doi: 10.1371/journal.pone.0097600

    Figure Lengend Snippet: Determination of expression level of LdTryS in sensitive (S) vs. drug resistant (R) strains of L. donovani. (A) Semiquantitative RT-PCR analysis of LdTryS transcript in Amp B sensitive vs. resistant isolates. Ethidium bromide-stained PCR products were photographed and the image was analyzed densitometrically. α-tubulin was used as control to show uniform expression of a housekeeping gene in both Amp B sensitive and resistant promastigotes. (B) Bar graph repr esents quantitative real time PCR analysis of LdTryS expression level in Amp B sensitive vs. resistant isolates. Data are normalized by the target/reference ratio of the calibrator. (C) The total Leishmania lysates (30 µg) were electrophoresed on 10% SDS-PAGE gel and stained with coomassie brilliant blue. Lane 1 represents, protein marker; lane 2 represents, sensitive strain (S); lanes 3, and 4 represent, resistant isolates (R1, R2). (D) Shows western blot of same coomassie gel using anti-LdTryS (1∶3000). The image was analyzed by densitometrically. Data was normalized and β-actin was used as control. The experiments were repeated twice and graphs represent the mean ± SD.

    Article Snippet: Digitonin Fractionation of L. donovani Promastigotes The differential membrane permeabilization of L. donovani promastigotes was done using digitonin (Calbiochem), as described previously , .

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, SDS Page, Marker, Western Blot

    Effect of H 2 O 2 on growth inhibition of L. donovani parasites. (A) L. donovani promastigotes (1×10 6 cells/ml) culture was treated with increasing concentration of H 2 O 2 (0–200 µM) up to 15 hrs and growth inhibitory effect of H 2 O 2 determined by MTT assay at 3 hr intervals. The cell viability after exposure with increasing concentration of H 2 O 2 was determined to optimize time of exposure and dose. (B) The intracellular ROS level was determined by quantification of DCF fluorescence. Results were normalized with cell numbers and presented relative to untreated control cells. (C) To confirm intracellular ROS production a quenching study was performed. The parasites treated with H 2 O 2 in the presence of 20 µM N -acetyl-L-cysteine (NAC) ROS scavenger reversed the effect of H 2 O 2. The experiments were repeated three times and graphs represent the mean ± SD.

    Journal: PLoS ONE

    Article Title: Stage-Dependent Expression and Up-Regulation of Trypanothione Synthetase in Amphotericin B Resistant Leishmania donovani

    doi: 10.1371/journal.pone.0097600

    Figure Lengend Snippet: Effect of H 2 O 2 on growth inhibition of L. donovani parasites. (A) L. donovani promastigotes (1×10 6 cells/ml) culture was treated with increasing concentration of H 2 O 2 (0–200 µM) up to 15 hrs and growth inhibitory effect of H 2 O 2 determined by MTT assay at 3 hr intervals. The cell viability after exposure with increasing concentration of H 2 O 2 was determined to optimize time of exposure and dose. (B) The intracellular ROS level was determined by quantification of DCF fluorescence. Results were normalized with cell numbers and presented relative to untreated control cells. (C) To confirm intracellular ROS production a quenching study was performed. The parasites treated with H 2 O 2 in the presence of 20 µM N -acetyl-L-cysteine (NAC) ROS scavenger reversed the effect of H 2 O 2. The experiments were repeated three times and graphs represent the mean ± SD.

    Article Snippet: Digitonin Fractionation of L. donovani Promastigotes The differential membrane permeabilization of L. donovani promastigotes was done using digitonin (Calbiochem), as described previously , .

    Techniques: Inhibition, Concentration Assay, MTT Assay, Fluorescence

    Subcellular localization of LdTryS. (A) Differential digitonin permeabilization of stationary phase promastigotes with increasing concentrations of digitonin. Supernatant and pellet fractions were run on 10% SDS-PAGE and transferred on to nitrocellulose membrane for western blot analysis using anti-LdTryS (1∶3000), anti LdcTXN (1∶4000), and anti LdIscS (1∶2000). cTXN and IscS served as cytosolic and mitochondrial markers, respectively. (B) Immunofluorescence microscopy of L. donovani promastigote with anti-LdTryS sera: phase contrast image, DAPI stained nucleus (N) and kinetoplast (K), Mitotracker stained mitochondria, anti-TryS labeled promastigote along with its merged image with DAPI is showing TryS localization in the cytoplasm.

    Journal: PLoS ONE

    Article Title: Stage-Dependent Expression and Up-Regulation of Trypanothione Synthetase in Amphotericin B Resistant Leishmania donovani

    doi: 10.1371/journal.pone.0097600

    Figure Lengend Snippet: Subcellular localization of LdTryS. (A) Differential digitonin permeabilization of stationary phase promastigotes with increasing concentrations of digitonin. Supernatant and pellet fractions were run on 10% SDS-PAGE and transferred on to nitrocellulose membrane for western blot analysis using anti-LdTryS (1∶3000), anti LdcTXN (1∶4000), and anti LdIscS (1∶2000). cTXN and IscS served as cytosolic and mitochondrial markers, respectively. (B) Immunofluorescence microscopy of L. donovani promastigote with anti-LdTryS sera: phase contrast image, DAPI stained nucleus (N) and kinetoplast (K), Mitotracker stained mitochondria, anti-TryS labeled promastigote along with its merged image with DAPI is showing TryS localization in the cytoplasm.

    Article Snippet: Digitonin Fractionation of L. donovani Promastigotes The differential membrane permeabilization of L. donovani promastigotes was done using digitonin (Calbiochem), as described previously , .

    Techniques: SDS Page, Western Blot, Immunofluorescence, Microscopy, Staining, Labeling