rabbit polyclonal anti β lactamase antibody  (Merck & Co)

Journal: Infection and Immunity

Article Title: Role of Yersinia pestis Toxin Complex Family Proteins in Resistance to Phagocytosis by Polymorphonuclear Leukocytes

doi: 10.1128/IAI.00648-13

Figure Lengend Snippet: T3SS-dependent secretion of the Y. pestis effector protein YopM but not the Tc proteins YitA and YipA. Y. pestis KIM10(pUC19::yitR) and Y. pestis(pMM83) (expresses YopM–β-lactamase) with or without pCD1::kan (T3SS+ or T3SS−) were grown in TMH overnight at 21°C, normalized, and then subcultured into TMH with or without CaCl2 and incubated at either 21°C or 37°C for an additional 4 h. Concentrated culture supernatants were analyzed by Western blotting using anti-YitA, anti-YipA, and anti-β-lactamase antibodies. Anti-GroEL was used to evaluate cellular lysis. Results are representative of three separate experiments.

Article Snippet: Proteins fused with β-lactamase were detected using a rabbit anti-β-lactamase polyclonal antibody (Millipore).

Techniques: Incubation, Western Blot, Lysis

Journal: Infection and Immunity

Article Title: Role of Yersinia pestis Toxin Complex Family Proteins in Resistance to Phagocytosis by Polymorphonuclear Leukocytes

doi: 10.1128/IAI.00648-13

Figure Lengend Snippet: Detection of T3SS-dependent secretion of YopE– and YopM–β-lactamase but not YitA– or YipA–β-lactamase in culture supernatants. Y. pestis(pCR-XL-TOPO::yitR) strains with (KIM5) or without (KIM6) the pCD1 virulence plasmid encoding the T3SS and engineered to express YitA–, YipA–, YopM– (pMM83), or YopE–β-lactamase (pMM85) were grown in TMH overnight at 21°C, normalized, and then subcultured in TMH (without CaCl2) and incubated at 21°C (gray columns) or 37°C (black columns) for an additional 4 h. Bacterial lysates (A) and culture supernatants (B) were incubated with fluorocillin for 4 h, and the fluorescence of each sample was measured. The means and standard errors for three experiments are indicated. Y. pestis KIM5 lacking β-lactamase fusion proteins and sterile TMH medium were used as background controls.

Article Snippet: Proteins fused with β-lactamase were detected using a rabbit anti-β-lactamase polyclonal antibody (Millipore).

Techniques: Plasmid Preparation, Incubation, Fluorescence, Sterility

Journal: Infection and Immunity

Article Title: Role of Yersinia pestis Toxin Complex Family Proteins in Resistance to Phagocytosis by Polymorphonuclear Leukocytes

doi: 10.1128/IAI.00648-13

Figure Lengend Snippet: YopM– and YopE–β-lactamase are translocated into CHO cells and PMNs via the T3SS, but YitA– and YipA–β-lactamase are not. (A) Representative images of CHO cells and human PMNs that were loaded with the fluorescent β-lactamase substrate CCF2-AM and incubated with Y. pestis KIM5(pMM85), expressing YopE–β-lactamase, KIM5(pCR-XL-TOPO::yitR), expressing YitA–β-lactamase, or KIM5(pCR-XL-TOPO::yitR), expressing YipA–β-lactamase. Intact CCF2-AM fluoresces green, and β-lactamase-cleaved CCF2-AM fluoresces blue. The cumulative flow cytometry results for three separate experiments are shown as the percentages of CHO cells (B) and PMNs (C) that contained cleaved CCF2-AM following incubation with Y. pestis with (KIM5) or without (KIM6) the T3SS-encoding pCD1 and expressing either YitA–, YipA–, YopE–, or YopM–β-lactamase. Uninfected cells or cells incubated with KIM5 lacking β-lactamase fusion proteins were used as negative controls. The means and standard errors are indicated. Data were analyzed by one-way ANOVA with the Bonferroni posttest. *, P < 0.05 [KIM5(pMM85) and KIM5(pMM83) compared to the other strains tested].

Article Snippet: Proteins fused with β-lactamase were detected using a rabbit anti-β-lactamase polyclonal antibody (Millipore).

Techniques: Incubation, Expressing, Flow Cytometry

Journal:

Article Title: RhuR, an Extracytoplasmic Function Sigma Factor Activator, Is Essential for Heme-Dependent Expression of the Outer Membrane Heme and Hemoprotein Receptor of Bordetella avium

doi: 10.1128/IAI.72.2.896-907.2004

Figure Lengend Snippet: The N terminus of RhuR activates PbhuR in the absence of hemin. Fe-stressed overnight cultures were used to inoculate high-Fe (BHI plus 150 μM FeSO4), low-Fe (BHI plus 100 μM EDDHA), and low-Fe plus hemin (BHI plus 100 μM EDDHA plus 1 μM hemin) broth cultures. After 16 h of incubation, the β-galactosidase activity of the cultures was determined using a modified Miller assay (27). (A) Activity of the RhuR fusions in wt B. avium. pAEK21 expresses a chimeric protein consisting of the first 97 amino acids of RhuR fused to β-lactamase (BlaM); pAEK23 encodes the same 97-amino-acid region of RhuR fused to 18 irrelevant amino acids encoded by the vector. (B) PbhuR promoter activity of the RhuR fusions in 4169rifΔrhuR. Numbers represent the average β-galactosidase activity of triplicate cultures. Error bars indicate ±1 standard deviation. Asterisks indicate a significant difference (P < 0.05) from the vector control cultured under the same condition, as determined by Student's t test.

Article Snippet: Rabbit polyclonal anti-β-lactamase antibodies (5 Prime-3 Prime, Inc., Boulder, Colo.) and peroxidase-conjugated anti-rabbit immunoglobulin G (whole molecule; Sigma Biochemicals) antibodies were used to detect RhuR-BlaM fusion protein.

Techniques: Incubation, Activity Assay, Modification, Plasmid Preparation, Standard Deviation, Cell Culture

Journal:

Article Title: RhuR, an Extracytoplasmic Function Sigma Factor Activator, Is Essential for Heme-Dependent Expression of the Outer Membrane Heme and Hemoprotein Receptor of Bordetella avium

doi: 10.1128/IAI.72.2.896-907.2004

Figure Lengend Snippet: RhuR-BlaM is localized to the inner membrane of 4169rifΔrhuR. Fe-stressed cultures of 4169rifΔrhuR(pDJM41, pRK415) (vector) and 4169rifΔrhuR(pDJM41, pAEK21) (RhuR-BlaM) were used to inoculate BHI broth supplemented with 100 μM EDDHA and 1 μM hemin. At stationary phase, cells were collected for isolation of total membranes, outer membranes, and cytoplasmic membranes. Ten micrograms of total protein from each membrane preparation was separated by SDS-PAGE using SDS-12% polyacrylamide gels. Western immunoblotting using a rabbit polyclonal anti-BlaM antiserum was used to detect immunoreactive proteins. The positions of the molecular mass markers (in kilodaltons) are indicated. Arrows indicate the position of the 37-kDa RhuR-BlaM and its major 25-kDa degradation product.

Article Snippet: Rabbit polyclonal anti-β-lactamase antibodies (5 Prime-3 Prime, Inc., Boulder, Colo.) and peroxidase-conjugated anti-rabbit immunoglobulin G (whole molecule; Sigma Biochemicals) antibodies were used to detect RhuR-BlaM fusion protein.

Techniques: Plasmid Preparation, Isolation, SDS Page, Western Blot