Bacterial Toxin Research Citations

In vitro protease assays:

Purified InhA1 was mixed with the substrate in protease buffer (50 mM Tris, 100 mM NaCl, 1 mM CaCl₂, 1 mM MgSO₄ [pH 7.8]) and incubated at 37C for set time points. Reaction mixtures were then placed on ice, and EDTA was added to a final concentration of 50 mM to stop the reaction. SDS buffer (2) was added, and samples were boiled for 5 min, separated by SDS-PAGE, and analyzed by Western blotting with substrate-specific antibodies. Recombinant PA (rPA), rLF, and rSodA-1 from B. anthracis were obtained from BEI Resources (ATCC, Manassas, VA). rEF was obtained from List Biological Laboratories (Campbell, CA). rNpr599 was purified as described above.

Author did not specify which List Labs Anthrax Edema Factor was utilized.  List Labs provides Product #178A(Anthrax Edema Factor (EF), Recombinant from B. anthracis) and Product #173B (Anthrax Edema Factor (EF), Recombinant, Mutant S447N from B. anthracis).

Supernatant ELISAs and LDH assays:

…Cells were infected at a multiplicity of 3:1, 5:1, or 10:1 (parasite to host cells) for various times. ATP (5 mM; Invivogen) and lethal toxin, composed of 1 g/ml each lethal factor (LF) and protective antigen (List Biological Laboratories), were used as controls. …

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Screening Against LF:

… Each well contained a volume of 90 L including buffer (20 mM HEPES, pH 7.4), recombinant LF (10 nM final concentration, List Biological Laboratories), and the fragment solution (1 mM final concentration). After a 20 min incubation period at 25 C, the reaction was initiated by the addition of 10 L fluorogenic LF substrate (2 M final concentration, MAPKKide DABCYL/FITC, List Biological Laboratories).

Author did not specify which Lethal Factor (LF) was utilized; List Labs provides the following LF:

• Prodcut #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence

Cytotoxicity studies:  Recombinant B. anthracis lethal factor was purchased from List Biological Laboratories (Campbell, CA, USA). …

Author did not specify which List Labs Lethal Factor was utilized.  List Labs offers Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

… B. anthracis rPA protein was from List Biological Laboratories, Inc. (Campbell, CA). …

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Materials and Methods:

… Recombinant protective antigen (rPA) of recent production from List Biological Laboratories Inc. was used as a control for these studies.

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

… Anthrax protective antigen-83 (PA83) was purchased from List Biological Laboratories. …

MPII Proteolysis of Proteins in Vitro:

Anthrax PA83 (2 g; 1 m) was co-incubated for 1 h at 37 C with increasing concentrations of MPII in 50 mm HEPES, pH 8.0, containing 1 mm CaCl₂, 0.5 mm MgCl₂, and 10 m ZnCl₂. The total volume of the reactions was 20 l. Where indicated, GM6001 (1 m) was added to the reactions to inhibit MPII. The cleavage reaction was stopped by adding a 5 SDS sample buffer. The digest samples were analyzed by SDS-PAGE using 420% polyacrylamide gel.

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Chemicals and Reagents:

PA and LF were purchased from List Biological Laboratories…Batches of PA and LF had different potency, accounting for the different amounts used in different experiments, each of which included control data for the same toxin batch.

Toxin Treatment and Cell-Viability Assay:

Cells were seeded in a 96-well plate at a density of 2 10⁵ cells/mL 1 d before toxin treatment. Various concentrations of PA combined with a fixed concentration of LF (200 ng/mL) were added to the wells, and cells were incubated for 4 h at 37 C.

Immunofluorescence Microscopy.

PA protein was labeled with Alexa-Fluor 488 using the protein-labeling kit…

Biochemical Assay of PA Binding and Internalization.

Cells were exposed to 1 g/mL of PA at 4 C for 1 h for the binding assay and then were shifted to 37 C for the indicated time for the internalization kinetics assay.

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

LT treatment of NRVM:

Approximately 24 h after NRVM plating, NRVM were treated with LT in a 2:1 ratio of PA:LF prepared in dispersion medium. PA (activated, 63 kD segment) and LF were purchased from List Biological Laboratories (Campbell, CA) and each reconstituted in glycerol at a concentration of 0.5 mg/mL.

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Human cytokine antibody array assay: 

After separation by centrifugation, WBCs were washed twice with complete RPMI 1640 medium, and the cells were resuspended in RPMI 1640 supplemented with 10% fetal bovine serum and penicillin-streptomycin. WBCs (5 10⁶ to 10 10⁶ cells per ml) were treated with ET (25 pmol PA plus 25 pmol EF), LT (25 pmol PA plus 25 pmol LF), …

The following materials were used in this study. B. anthracis EF, LF, and PA were purchased from List Biological Laboratories (Campbell, CA).

Author did not indicate which specific lethal factor was utilized.  List Labs provides Product #172 (Anthrax Lethal Factor (LF), Recombinant from B. anthracis) and Product #169 (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).

• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Author did not specify which List Labs Anthrax Edema Factor was utilized.  List Labs provides Product #178A(Anthrax Edema Factor (EF), Recombinant from B. anthracis) and Product #173B (Anthrax Edema Factor (EF), Recombinant, Mutant S447N from B. anthracis).