p7719  (New England Biolabs)

Journal: PLoS ONE

Article Title: Generation of an artificial human B cell line test system using Transpo-mAbTM technology to evaluate the therapeutic efficacy of novel antigen-specific fusion proteins

doi: 10.1371/journal.pone.0180305

Figure Lengend Snippet: Expression and characterization of TTC-based fusions proteins. (A) The TTC DNA sequence was transferred to the pMS vector system using the SfiI/NotI sites to generate pMS-EGrB(R201K)-TTC and pMS-TTC-MAPTau. Abbreviations: CMV = cytomegalovirus promoter, Ig kappa = murine signal sequence for protein secretion into the cell culture supernatant, ECS = enterokinase cleavage site, His 10 = polyhistidine tag, IRES = internal ribosome entry site for the co-expression of eGFP, eGFP = enhanced green fluorescent protein. (B) EGrB(R201K)-TTC and TTC-MAPTau were expressed in HEK 293T cells and purified by IMAC. The GrB-(R210K)-TTC and TTC-MAPTau proteins were separated by denaturing SDS-PAGE followed by staining with Coomassie Brilliant Blue (left). Western blot analysis (right) using anti-polyhistidine and goat anti-mouse IgG (Fc specific) antibodies revealed protein bands of the anticipated sizes for GrBR201K-TTC (80 kDa) and TTC-MAPTau (93 kDa). Lane 1—Color Prestained Protein Standard, Broad Range (11–245 k); lane 2—GrBR201K-TTC, lane 3—TTC-MAPTau. (C) EGrB(R201K)-TTC was digested with enterokinase and granzyme B activity was tested using substrate Ac-IETD- p NA (white circle) compared to uncleaved EGrB(R201K)-TTC (black triangle) and the mock-protein (white triangle). The enzymatic activity of the granzyme B domain was determined using a colorimetric assay and the absorbance at 405 nm was monitored for 60 min in 2-min intervals. (D) An XTT-based cell viability assay was carried out using serial dilutions of the novel TTC-fusion proteins against the mouse TTC-reactive hybridoma cell line 5E4 (72 h, 37°C, 5% CO 2 ). The data are means ± standard deviation (SD) of technical triplicates of three independent experiments (n = 3).

Article Snippet: Purified proteins were separated by SDS-PAGE and visualized by Coomassie Brilliant Blue staining against Color Prestained Protein Standards, Broad Range (New England Biolabs, Ipswich, Massachusetts).

Techniques: Expressing, Sequencing, Plasmid Preparation, Cell Culture, Purification, SDS Page, Staining, Western Blot, Activity Assay, Colorimetric Assay, Viability Assay, Standard Deviation

Journal: Life

Article Title: Cloning, Expression and Characterization of UDP-Glucose Dehydrogenases

doi: 10.3390/life11111201

Figure Lengend Snippet: SDS–PAGE gel showing 6-His-tagged uridine diphosphate-glucose dehydrogenases (UGDs) expression in Escherichia coli BL21. Lane L on the left corresponds to the ladder Color Prestained Protein Standard, Broad Range (11–245 kDa). Samples loaded were: ( 1 ) soluble cell extract of E. coli pRSFDuet-1 (15 µg), ( 2 ) soluble cell extract from E. coli pRSFDuet_ Zm UGD (15 µg), ( 3 ) 1st eluted fraction from Zm UGD purification (5 µg), ( 4 ) soluble cell extract from E. coli pRSFDuet_ Lbj UGD (15 µg), ( 5 ) 1st eluted fraction from Lbj UGD purification (5 µg), ( 6,7 ) 2nd eluted fractions from Zm UGD and Lbj UGD purification, respectively (5 µg).

Article Snippet: The protein marker used was Color Protein Standard—Broad Range (NEB, #77125) or NZYColour Protein Marker II (NZYTech).

Techniques: SDS Page, Expressing, Purification

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

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

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:

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:

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

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

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