whia  (New England Biolabs)


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    Name:
    Blue Prestained Protein Standard Broad Range 11 190 kDa
    Description:
    Blue Prestained Protein Standard Broad Range 11 190 kDa 750 mini gel lanes
    Catalog Number:
    P7706L
    Price:
    576
    Category:
    Protein Molecular Weight Markers
    Size:
    750 mini gel lanes
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    Structured Review

    New England Biolabs whia
    Blue Prestained Protein Standard Broad Range 11 190 kDa
    Blue Prestained Protein Standard Broad Range 11 190 kDa 750 mini gel lanes
    https://www.bioz.com/result/whia/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    whia - by Bioz Stars, 2021-09
    94/100 stars

    Images

    1) Product Images from "Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis Shows that WhiB Is a Transcription Factor That Cocontrols Its Regulon with WhiA To Initiate Developmental Cell Division in Streptomyces"

    Article Title: Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis Shows that WhiB Is a Transcription Factor That Cocontrols Its Regulon with WhiA To Initiate Developmental Cell Division in Streptomyces

    Journal: mBio

    doi: 10.1128/mBio.00523-16

    WhiA and WhiB have a shared regulon. The data compare anti-FLAG ChIP-seq results for WhiA and WhiB. ChIP traces are shown for 12 selected WhiA and WhiB target genes: ftsW , ftsZ , filP , whiG , sven1406 , sven5313 , sven3535 , sven4724 , sven1324 , sven4756 , sven6616 , and sven3229 . Color coding of the ChIP samples is as follows: 3×FLAG-[Gly 4 Ser] 3 -WhiB strain (WhiB-FLAG), red; corresponding S. venezuelae wild-type anti-FLAG negative control (WT), green; 3×FLAG-[Gly 4 Ser] 3 -WhiA strain (WhiA-FLAG), blue; and corresponding S. venezuelae wild-type anti-FLAG negative control (WT), purple. Plots span approximately 3 kb of DNA sequence. Genes running left to right are shown in green, and genes running right to left are shown in red. The black arrow indicates the gene subject to WhiA and WhiB regulation. The arrangement of the 12 panels mirrors that in Fig. 4 .
    Figure Legend Snippet: WhiA and WhiB have a shared regulon. The data compare anti-FLAG ChIP-seq results for WhiA and WhiB. ChIP traces are shown for 12 selected WhiA and WhiB target genes: ftsW , ftsZ , filP , whiG , sven1406 , sven5313 , sven3535 , sven4724 , sven1324 , sven4756 , sven6616 , and sven3229 . Color coding of the ChIP samples is as follows: 3×FLAG-[Gly 4 Ser] 3 -WhiB strain (WhiB-FLAG), red; corresponding S. venezuelae wild-type anti-FLAG negative control (WT), green; 3×FLAG-[Gly 4 Ser] 3 -WhiA strain (WhiA-FLAG), blue; and corresponding S. venezuelae wild-type anti-FLAG negative control (WT), purple. Plots span approximately 3 kb of DNA sequence. Genes running left to right are shown in green, and genes running right to left are shown in red. The black arrow indicates the gene subject to WhiA and WhiB regulation. The arrangement of the 12 panels mirrors that in Fig. 4 .

    Techniques Used: Chromatin Immunoprecipitation, Negative Control, Sequencing

    Mutation of the cysteine residues that coordinate the WhiB [4Fe-4S] cluster prevents DNA binding in vivo . Anti-FLAG ChIP-seq data for WhiB and WhiB (4C-S) are shown for three representative WhiA and WhiB target genes: ftsW , ftsZ , and filP . Color coding of the ChIP samples is as follows: 3×FLAG-[Gly 4 Ser] 3 -WhiB strain (WhiB-FLAG), red; 3×FLAG-[Gly 4 Ser] 3 -WhiB(4C-S) strain, green; corresponding S. venezuelae wild-type anti-FLAG negative control (WT), blue. Plots span approximately 3 kb of DNA sequence. Genes running right to left are shown in red. The black arrow indicates the gene subject to WhiA and WhiB regulation.
    Figure Legend Snippet: Mutation of the cysteine residues that coordinate the WhiB [4Fe-4S] cluster prevents DNA binding in vivo . Anti-FLAG ChIP-seq data for WhiB and WhiB (4C-S) are shown for three representative WhiA and WhiB target genes: ftsW , ftsZ , and filP . Color coding of the ChIP samples is as follows: 3×FLAG-[Gly 4 Ser] 3 -WhiB strain (WhiB-FLAG), red; 3×FLAG-[Gly 4 Ser] 3 -WhiB(4C-S) strain, green; corresponding S. venezuelae wild-type anti-FLAG negative control (WT), blue. Plots span approximately 3 kb of DNA sequence. Genes running right to left are shown in red. The black arrow indicates the gene subject to WhiA and WhiB regulation.

    Techniques Used: Mutagenesis, Binding Assay, In Vivo, Chromatin Immunoprecipitation, Negative Control, Sequencing

    Related Articles

    Fluorescence:

    Article Title: Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei
    Article Snippet: .. 5.4 Flavinylation analysis by in-gel fluorescence As described before [ ], SDS-PAGE gels were scanned with a Typhoon TRIO Variable Mode Imager System (GE Healthcare). λ ex = 488 nm and λ em = 526 nm were used for detection of covalently bound flavin and λ ex = 670 nm and λ em = 633 nm for visualisation of the Blue Prestained Protein Standard (New England Biolabs). ..

    SDS Page:

    Article Title: Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei
    Article Snippet: .. 5.4 Flavinylation analysis by in-gel fluorescence As described before [ ], SDS-PAGE gels were scanned with a Typhoon TRIO Variable Mode Imager System (GE Healthcare). λ ex = 488 nm and λ em = 526 nm were used for detection of covalently bound flavin and λ ex = 670 nm and λ em = 633 nm for visualisation of the Blue Prestained Protein Standard (New England Biolabs). ..

    Article Title: Metabolic selection of a homologous recombination mediated loss of glycosomal fumarate reductase in Trypanosoma brucei
    Article Snippet: .. Analysis of FRDg flavinylationAs described in , gels resulting from SDS-PAGE were scanned with a Typhoon TRIO Variable Mode Imager System (GE Healthcare) at λex = 488 nm and λem = 526 nm for detection of covalently bound flavin and at λex = 670 nm and λem = 633 nm for visualization of the Blue Prestained Protein Standard (NEB). ..

    Article Title: Metabolic selection of a homologous recombination-mediated gene loss protects Trypanosoma brucei from ROS production by glycosomal fumarate reductase
    Article Snippet: .. Analysis of FRDg flavinylationAs described in , gels resulting from SDS-PAGE were scanned with a Typhoon TRIO Variable Mode Imager System (GE Healthcare) at λex = 488 nm and λem = 526 nm for detection of covalently bound flavin and at λex = 670 nm and λem = 633 nm for visualization of the Blue Prestained Protein Standard (NEB). ..

    Marker:

    Article Title: Redirected nuclear glutamate dehydrogenase supplies Tet3 with α-ketoglutarate in neurons
    Article Snippet: .. Samples were loaded on a 4–15% precast polyacrylamide gel (Bio-Rad) and MagicMark XP Standard (Thermo Fisher LC5603) and Blue Prestained Protein Standard, Broad Range (11–190 kDa) (New England Biolabs P7706S) or Color-coded Prestained Protein Marker, Broad Range (11–250 kDa) (New England Biolabs 14208) were used as protein standards. ..

    Molecular Weight:

    Article Title: Increased circulating levels of Factor H-Related Protein 4 are strongly associated with age-related macular degeneration
    Article Snippet: .. Molecular weight markers used were Blue Prestained Protein Standards, Broad Range (11–190 kDa, New England BioLabs, Hitchin, UK, catalogue no. P7706S). ..

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    New England Biolabs prestained protein markers
    Production of extracellular and cell-bound CPSs by C. jejuni grown on minimal medium. (A and B) The gel was stained with either Alcian blue (A) or Alcian blue and silver (B). Lanes 1, 3, 5, and 7, wild-type strain; lanes 2, 4, 6, and 8, kpsM mutant (control); lanes 1 to 6, culture supernatant; lanes 7 and 8, total cell lysate; lanes 1 and 2, no DOC present; lanes 3 and 4, DOC present in the growth medium; lanes 4 and 5, DOC added 3 h before the end of incubation. A sharp ∼18-kDa band is present in all lanes in panel B and comigrates with CPS-PL, corresponding to proteinase K. (C) Effect of phospholipases on the product (CPS) accumulated in the supernatant in the presence of DOC (from panel A, lane 3). Lane 1, CPS-PL (control); lane 2, CPS; lanes 3 and 4, limited and complete hydrolysis with PL, respectively; lane M, <t>prestained</t> markers.
    Prestained Protein Markers, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Production of extracellular and cell-bound CPSs by C. jejuni grown on minimal medium. (A and B) The gel was stained with either Alcian blue (A) or Alcian blue and silver (B). Lanes 1, 3, 5, and 7, wild-type strain; lanes 2, 4, 6, and 8, kpsM mutant (control); lanes 1 to 6, culture supernatant; lanes 7 and 8, total cell lysate; lanes 1 and 2, no DOC present; lanes 3 and 4, DOC present in the growth medium; lanes 4 and 5, DOC added 3 h before the end of incubation. A sharp ∼18-kDa band is present in all lanes in panel B and comigrates with CPS-PL, corresponding to proteinase K. (C) Effect of phospholipases on the product (CPS) accumulated in the supernatant in the presence of DOC (from panel A, lane 3). Lane 1, CPS-PL (control); lane 2, CPS; lanes 3 and 4, limited and complete hydrolysis with PL, respectively; lane M, prestained markers.

    Journal: Journal of Clinical Microbiology

    Article Title: Detection and Initial Characterization of Novel Capsular Polysaccharide among Diverse Campylobacter jejuni Strains Using Alcian Blue Dye

    doi: 10.1128/JCM.39.1.279-284.2001

    Figure Lengend Snippet: Production of extracellular and cell-bound CPSs by C. jejuni grown on minimal medium. (A and B) The gel was stained with either Alcian blue (A) or Alcian blue and silver (B). Lanes 1, 3, 5, and 7, wild-type strain; lanes 2, 4, 6, and 8, kpsM mutant (control); lanes 1 to 6, culture supernatant; lanes 7 and 8, total cell lysate; lanes 1 and 2, no DOC present; lanes 3 and 4, DOC present in the growth medium; lanes 4 and 5, DOC added 3 h before the end of incubation. A sharp ∼18-kDa band is present in all lanes in panel B and comigrates with CPS-PL, corresponding to proteinase K. (C) Effect of phospholipases on the product (CPS) accumulated in the supernatant in the presence of DOC (from panel A, lane 3). Lane 1, CPS-PL (control); lane 2, CPS; lanes 3 and 4, limited and complete hydrolysis with PL, respectively; lane M, prestained markers.

    Article Snippet: Prestained protein markers (New England BioLabs) were used.

    Techniques: Staining, Mutagenesis, Incubation

    Detection of CPS using Alcian blue in different C. jejuni strains. Lanes: 1, NCTC 12501 (HS:2); 2, NCTC 12502 (HS:3); 3, NCTC 12561 (HS:4); 4, NCTC 12509 (HS:10); 5, NCTC 12517 (HS:19); 6, 81116 (NCTC 11828, HS:6, 7); 7, NCTC 11168 (HS:2); 8, G1 (HS:1); 9, × (untypeable); 10, prestained markers.

    Journal: Journal of Clinical Microbiology

    Article Title: Detection and Initial Characterization of Novel Capsular Polysaccharide among Diverse Campylobacter jejuni Strains Using Alcian Blue Dye

    doi: 10.1128/JCM.39.1.279-284.2001

    Figure Lengend Snippet: Detection of CPS using Alcian blue in different C. jejuni strains. Lanes: 1, NCTC 12501 (HS:2); 2, NCTC 12502 (HS:3); 3, NCTC 12561 (HS:4); 4, NCTC 12509 (HS:10); 5, NCTC 12517 (HS:19); 6, 81116 (NCTC 11828, HS:6, 7); 7, NCTC 11168 (HS:2); 8, G1 (HS:1); 9, × (untypeable); 10, prestained markers.

    Article Snippet: Prestained protein markers (New England BioLabs) were used.

    Techniques:

    Analysis of CPS extracted from C. jejuni ) (D). Lanes 1, supernatant of cells washed with saline; lanes 2, 50°C extract; lanes 3, lysate; lane 4, prestained markers.

    Journal: Journal of Clinical Microbiology

    Article Title: Detection and Initial Characterization of Novel Capsular Polysaccharide among Diverse Campylobacter jejuni Strains Using Alcian Blue Dye

    doi: 10.1128/JCM.39.1.279-284.2001

    Figure Lengend Snippet: Analysis of CPS extracted from C. jejuni ) (D). Lanes 1, supernatant of cells washed with saline; lanes 2, 50°C extract; lanes 3, lysate; lane 4, prestained markers.

    Article Snippet: Prestained protein markers (New England BioLabs) were used.

    Techniques:

    Thermal stability of the C. jejuni G1 CPS. Western blotting with Penner 1 antisera of fraction F2 (see text) after incubation at 100°C for 0 (lane 1), 5 (lane 2), and 10 (lane 3) min. Lane 4 shows prestained markers.

    Journal: Journal of Clinical Microbiology

    Article Title: Detection and Initial Characterization of Novel Capsular Polysaccharide among Diverse Campylobacter jejuni Strains Using Alcian Blue Dye

    doi: 10.1128/JCM.39.1.279-284.2001

    Figure Lengend Snippet: Thermal stability of the C. jejuni G1 CPS. Western blotting with Penner 1 antisera of fraction F2 (see text) after incubation at 100°C for 0 (lane 1), 5 (lane 2), and 10 (lane 3) min. Lane 4 shows prestained markers.

    Article Snippet: Prestained protein markers (New England BioLabs) were used.

    Techniques: Western Blot, Incubation

    Gel detection of C. jejuni G1 CPS (lane 1) and its lipid-free form CPS-PL (lane 2) by a combination of Alcian blue-silver staining procedures. S. enterica serovar Typhimurium LPS is present in lane 3 for comparison. Lane 4 shows prestained markers.

    Journal: Journal of Clinical Microbiology

    Article Title: Detection and Initial Characterization of Novel Capsular Polysaccharide among Diverse Campylobacter jejuni Strains Using Alcian Blue Dye

    doi: 10.1128/JCM.39.1.279-284.2001

    Figure Lengend Snippet: Gel detection of C. jejuni G1 CPS (lane 1) and its lipid-free form CPS-PL (lane 2) by a combination of Alcian blue-silver staining procedures. S. enterica serovar Typhimurium LPS is present in lane 3 for comparison. Lane 4 shows prestained markers.

    Article Snippet: Prestained protein markers (New England BioLabs) were used.

    Techniques: Silver Staining

    Effect of phospholipase treatment on binding of CPS to a Hybond N membrane. After blotting, the membrane was stained with Alcian blue as described in the text. (A) Lane 1, CPS; lane 2, prestained markers. (B) Lane 1, CPS-PL; lane 2, prestained markers.

    Journal: Journal of Clinical Microbiology

    Article Title: Detection and Initial Characterization of Novel Capsular Polysaccharide among Diverse Campylobacter jejuni Strains Using Alcian Blue Dye

    doi: 10.1128/JCM.39.1.279-284.2001

    Figure Lengend Snippet: Effect of phospholipase treatment on binding of CPS to a Hybond N membrane. After blotting, the membrane was stained with Alcian blue as described in the text. (A) Lane 1, CPS; lane 2, prestained markers. (B) Lane 1, CPS-PL; lane 2, prestained markers.

    Article Snippet: Prestained protein markers (New England BioLabs) were used.

    Techniques: Binding Assay, Staining

    Mutations in the apolar patch cause the prl phenotype. (A) Translocation assays of sec61 L66 mutants. Translocation of CPY and integration of DPAPB was assayed by 7-min pulse labeling of wild-type and mutant yeast cells. CPY and DPAPB were immunoprecipitated from pulse-labeled cell extracts using CPY- and DPAPB-specific antisera. The glycosylated ER forms of CPY (p1) and DPAPB were resolved from nontranslocated precursors (ppCPY and pDPAPB) by SDS-PAGE. The p1CPY doublet is caused by p1CPY glycoforms that have three or four N-linked glycans. The percentage of translocation (CPY) or integration (DPAPB) is the mean of four or more determinations, one of which is shown here. (B) The prl phenotype of sec61 mutants was assayed by pulse labeling using the ppCPYΔ2-T7 (Δ2) and ppCPYΔ4-T7 (Δ4) reporters. Precursors (ppCPYΔ2-T7 or ppCPYΔ4-T7) and the translocated product (p1CPY-T7) that were immunoprecipitated using anti-T7 antisera were resolved by SDS-PAGE. The percentage of translocation is the mean of four determinations; error bars designate standard deviations. (C) The Δ translocation value for a sec61 allele corresponds to the percentage of increase or decrease in p1CPY relative to wild-type Sec61 for the ppCPY, ppCPYΔ2, and ppCPYΔ4 reporters. Wild-type and mutant strains were all assayed two or more times. Table S1 includes the assay values (CPY translocation, DPAPB integration, and prl reporter translocation) and Δ translocation value for all mutants that are displayed in this panel. The error bars correspond to the sum of the individual errors for the three assays that were used to calculate the Δ translocation value. The indicated molecular masses shown in this and subsequent figures are the apparent molecular masses of the observed protein species relative to prestained molecular mass markers that were electrophoresed on all gels.

    Journal: The Journal of Cell Biology

    Article Title: A gating motif in the translocation channel sets the hydrophobicity threshold for signal sequence function

    doi: 10.1083/jcb.201207163

    Figure Lengend Snippet: Mutations in the apolar patch cause the prl phenotype. (A) Translocation assays of sec61 L66 mutants. Translocation of CPY and integration of DPAPB was assayed by 7-min pulse labeling of wild-type and mutant yeast cells. CPY and DPAPB were immunoprecipitated from pulse-labeled cell extracts using CPY- and DPAPB-specific antisera. The glycosylated ER forms of CPY (p1) and DPAPB were resolved from nontranslocated precursors (ppCPY and pDPAPB) by SDS-PAGE. The p1CPY doublet is caused by p1CPY glycoforms that have three or four N-linked glycans. The percentage of translocation (CPY) or integration (DPAPB) is the mean of four or more determinations, one of which is shown here. (B) The prl phenotype of sec61 mutants was assayed by pulse labeling using the ppCPYΔ2-T7 (Δ2) and ppCPYΔ4-T7 (Δ4) reporters. Precursors (ppCPYΔ2-T7 or ppCPYΔ4-T7) and the translocated product (p1CPY-T7) that were immunoprecipitated using anti-T7 antisera were resolved by SDS-PAGE. The percentage of translocation is the mean of four determinations; error bars designate standard deviations. (C) The Δ translocation value for a sec61 allele corresponds to the percentage of increase or decrease in p1CPY relative to wild-type Sec61 for the ppCPY, ppCPYΔ2, and ppCPYΔ4 reporters. Wild-type and mutant strains were all assayed two or more times. Table S1 includes the assay values (CPY translocation, DPAPB integration, and prl reporter translocation) and Δ translocation value for all mutants that are displayed in this panel. The error bars correspond to the sum of the individual errors for the three assays that were used to calculate the Δ translocation value. The indicated molecular masses shown in this and subsequent figures are the apparent molecular masses of the observed protein species relative to prestained molecular mass markers that were electrophoresed on all gels.

    Article Snippet: Molecular masses of protein products on SDS-PAGE gels were estimated relative to prestained molecular mass markers (Prestained Protein Marker, Broad Range; New England Biolabs, Inc.).

    Techniques: Translocation Assay, Labeling, Mutagenesis, Immunoprecipitation, SDS Page

    WhiA and WhiB have a shared regulon. The data compare anti-FLAG ChIP-seq results for WhiA and WhiB. ChIP traces are shown for 12 selected WhiA and WhiB target genes: ftsW , ftsZ , filP , whiG , sven1406 , sven5313 , sven3535 , sven4724 , sven1324 , sven4756 , sven6616 , and sven3229 . Color coding of the ChIP samples is as follows: 3×FLAG-[Gly 4 Ser] 3 -WhiB strain (WhiB-FLAG), red; corresponding S. venezuelae wild-type anti-FLAG negative control (WT), green; 3×FLAG-[Gly 4 Ser] 3 -WhiA strain (WhiA-FLAG), blue; and corresponding S. venezuelae wild-type anti-FLAG negative control (WT), purple. Plots span approximately 3 kb of DNA sequence. Genes running left to right are shown in green, and genes running right to left are shown in red. The black arrow indicates the gene subject to WhiA and WhiB regulation. The arrangement of the 12 panels mirrors that in Fig. 4 .

    Journal: mBio

    Article Title: Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis Shows that WhiB Is a Transcription Factor That Cocontrols Its Regulon with WhiA To Initiate Developmental Cell Division in Streptomyces

    doi: 10.1128/mBio.00523-16

    Figure Lengend Snippet: WhiA and WhiB have a shared regulon. The data compare anti-FLAG ChIP-seq results for WhiA and WhiB. ChIP traces are shown for 12 selected WhiA and WhiB target genes: ftsW , ftsZ , filP , whiG , sven1406 , sven5313 , sven3535 , sven4724 , sven1324 , sven4756 , sven6616 , and sven3229 . Color coding of the ChIP samples is as follows: 3×FLAG-[Gly 4 Ser] 3 -WhiB strain (WhiB-FLAG), red; corresponding S. venezuelae wild-type anti-FLAG negative control (WT), green; 3×FLAG-[Gly 4 Ser] 3 -WhiA strain (WhiA-FLAG), blue; and corresponding S. venezuelae wild-type anti-FLAG negative control (WT), purple. Plots span approximately 3 kb of DNA sequence. Genes running left to right are shown in green, and genes running right to left are shown in red. The black arrow indicates the gene subject to WhiA and WhiB regulation. The arrangement of the 12 panels mirrors that in Fig. 4 .

    Article Snippet: The positions and sizes of WhiA (red arrow and red asterisks) and 3×FLAG-[Gly4 Ser]3 -WhiA (blue arrow and blue asterisks) are indicated against an NEB (no. P7706) protein ladder.

    Techniques: Chromatin Immunoprecipitation, Negative Control, Sequencing

    Mutation of the cysteine residues that coordinate the WhiB [4Fe-4S] cluster prevents DNA binding in vivo . Anti-FLAG ChIP-seq data for WhiB and WhiB (4C-S) are shown for three representative WhiA and WhiB target genes: ftsW , ftsZ , and filP . Color coding of the ChIP samples is as follows: 3×FLAG-[Gly 4 Ser] 3 -WhiB strain (WhiB-FLAG), red; 3×FLAG-[Gly 4 Ser] 3 -WhiB(4C-S) strain, green; corresponding S. venezuelae wild-type anti-FLAG negative control (WT), blue. Plots span approximately 3 kb of DNA sequence. Genes running right to left are shown in red. The black arrow indicates the gene subject to WhiA and WhiB regulation.

    Journal: mBio

    Article Title: Genome-Wide Chromatin Immunoprecipitation Sequencing Analysis Shows that WhiB Is a Transcription Factor That Cocontrols Its Regulon with WhiA To Initiate Developmental Cell Division in Streptomyces

    doi: 10.1128/mBio.00523-16

    Figure Lengend Snippet: Mutation of the cysteine residues that coordinate the WhiB [4Fe-4S] cluster prevents DNA binding in vivo . Anti-FLAG ChIP-seq data for WhiB and WhiB (4C-S) are shown for three representative WhiA and WhiB target genes: ftsW , ftsZ , and filP . Color coding of the ChIP samples is as follows: 3×FLAG-[Gly 4 Ser] 3 -WhiB strain (WhiB-FLAG), red; 3×FLAG-[Gly 4 Ser] 3 -WhiB(4C-S) strain, green; corresponding S. venezuelae wild-type anti-FLAG negative control (WT), blue. Plots span approximately 3 kb of DNA sequence. Genes running right to left are shown in red. The black arrow indicates the gene subject to WhiA and WhiB regulation.

    Article Snippet: The positions and sizes of WhiA (red arrow and red asterisks) and 3×FLAG-[Gly4 Ser]3 -WhiA (blue arrow and blue asterisks) are indicated against an NEB (no. P7706) protein ladder.

    Techniques: Mutagenesis, Binding Assay, In Vivo, Chromatin Immunoprecipitation, Negative Control, Sequencing

    Cleavage of rhTPO by platelet suspensions. ( A ) Time course. Platelet pellets were resuspended in a nominally calcium-free modified Hepes⋅Tyrode buffer at a concentration of 3 × 10 8 platelets per ml. RhTPO (1 μg/ml) and 1 mM calcium ions were or were not added to the platelet suspension, and incubation was conducted for various times at 37°C. The platelets were then lysed by the addition of 2× concentrated SDS/PAGE buffer, and subjected to 7.5–15% gradient SDS/PAGE. Proteins were then electroblotted onto a PVDF membrane, stained by anti-rhTPO Ab, and detected by the ECL method, as described. Prestained protein markers were obtained from Amersham. Lanes: 1, platelet lysate only without exogenous TPO; 2, sample immediately lysed after the addition of rhTPO (1 μg/ml) and 1 mM calcium ions; 3–6, samples lysed 1, 10, 30, and 60 min, respectively, after those additions. The position of the intact rhTPO and the 34-kDa protein band of interest (∗) are indicated by the arrows. ( B ) The cleavage of rhTPO is inhibited by hirudin and requires calcium ions. Platelets were lysed by the addition of 2× concentrated SDS/PAGE buffer immediately or after incubation for 1 hr at 37°C with rhTPO (1 μg/ml) in the presence or absence of 1 mM calcium ions. Lysed samples were subjected to 7.5–15% gradient SDS/PAGE. RhTPO was detected as described in A . Lanes: 1, immediately lysed sample; 2, 1-hr incubation in the presence of calcium ion (1 mM); 3, 1-hr incubation without the addition of calcium ions; 4, 1-hr incubation with EGTA (1 mM); 5, 1-hr incubation in the presence of calcium ion plus hirudin (10 units per ml); 6, free rhTPO incubated with thrombin (10 units per ml).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Thrombin cleaves recombinant human thrombopoietin: One of the proteolytic events that generates truncated forms of thrombopoietin

    doi:

    Figure Lengend Snippet: Cleavage of rhTPO by platelet suspensions. ( A ) Time course. Platelet pellets were resuspended in a nominally calcium-free modified Hepes⋅Tyrode buffer at a concentration of 3 × 10 8 platelets per ml. RhTPO (1 μg/ml) and 1 mM calcium ions were or were not added to the platelet suspension, and incubation was conducted for various times at 37°C. The platelets were then lysed by the addition of 2× concentrated SDS/PAGE buffer, and subjected to 7.5–15% gradient SDS/PAGE. Proteins were then electroblotted onto a PVDF membrane, stained by anti-rhTPO Ab, and detected by the ECL method, as described. Prestained protein markers were obtained from Amersham. Lanes: 1, platelet lysate only without exogenous TPO; 2, sample immediately lysed after the addition of rhTPO (1 μg/ml) and 1 mM calcium ions; 3–6, samples lysed 1, 10, 30, and 60 min, respectively, after those additions. The position of the intact rhTPO and the 34-kDa protein band of interest (∗) are indicated by the arrows. ( B ) The cleavage of rhTPO is inhibited by hirudin and requires calcium ions. Platelets were lysed by the addition of 2× concentrated SDS/PAGE buffer immediately or after incubation for 1 hr at 37°C with rhTPO (1 μg/ml) in the presence or absence of 1 mM calcium ions. Lysed samples were subjected to 7.5–15% gradient SDS/PAGE. RhTPO was detected as described in A . Lanes: 1, immediately lysed sample; 2, 1-hr incubation in the presence of calcium ion (1 mM); 3, 1-hr incubation without the addition of calcium ions; 4, 1-hr incubation with EGTA (1 mM); 5, 1-hr incubation in the presence of calcium ion plus hirudin (10 units per ml); 6, free rhTPO incubated with thrombin (10 units per ml).

    Article Snippet: Prestained protein markers (New England BioLabs) were used for electrophoretic estimation of M r .

    Techniques: Modification, Concentration Assay, Incubation, SDS Page, Staining