Bacterial Toxin Research Citations

Serum samples from each mouse were then added to the monolayer of macrophages in increasing concentrations and serum and cells were co-cultured with PA (0.5 g/mL) and lethal factor (LF, 0.5 g/mL, List Biological Laboratories, Inc, 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

Enzyme-linked immunosorbent assay (ELISA):

Samples were grown overnight in LB-L media, then subcultured 1:100 in LB-L media and grown for 8 h at 37 C and 225 rpm. The cultures were pelleted by one centrifugation step of 2272g for 10 min and the supernatant was passed through a 0.45 m filter. The wells of a 96-well microtiter plate (Santa Cruz Biotechnology, Inc., part# sc-204463) was coated with 100 L of a solution of 4 g mL¹ protective antigen (PA) of the anthrax toxin (List Biological Laboratories, part# 171E) in 5 mM HEPES, 50 mM NaCl, pH 7.5 covered at 4 C overnight. Liquid was removed by inversion and wells were incubated with a 200 L blocking solution (200 L 2% milk, 0.05% TBST) at room temperature for 1 h. …

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

… anthrax lethal factor (batch 1692A1B), and protective antigen (batch 17117A1B) from List Biological Laboratories. …

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

List Labs Product #169A (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence; Lyophilized) has been discontinued; however, Product #169L (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence; Liquid) is available for purchase.

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

Materials:

… PA from B. anthracis in its holo form (PA83) and cleaved form (PA63) where purchased from List Biological Laboratories, …

In vitro selection scheme:

The AEGIS-LIVE experiment was performed on a synthetic library of GACTZP oligonucleotides containing 25 randomized positions flanked by two primer binding sites (59 nt in length, 5-AGAGAGCGTCGTGTGGA-N25-TGAGGAGGTGCGCAAGT-3).

PA was presented immobilized on magnetic beads…

Filter binding assays:

5-³²P-labeled aptamer (1 l each) was denatured/renatured in 1 PBS (30 l), and incubated (45 min, RT) with increasing concentrations of PA63 or PA83, from 0 to 12 M. To capture the PA and PA-aptamer complex, aliquots (5 l) from each binding reaction were spotted on a nitrocellulose membrane on top of a charged nylon membrane (Whatman) in a dot-blot minifold vacuum device (Millipore) (27). Membranes were washed …

Determination of the affinity constant with bead-based assay:

After being coupled to PA63 (1.6 nmoles), the beads (200 l, as during selection) were divided into aliquots (20 l each) and each fraction was incubated with increasing concentrations of …

Enzymatic footprinting:

For DNase I digestion of PA1 and PA1T4 in the presence of PA63 or PA83, the aptamer was denatured (85C, 5 min) followed by slow (0.1/s) cooling to 24C to allow refolding. 5³²P-PA1 with 05 M PA63 or PA83 was incubated with tumbling for 45 min, and then treated with DNase I nuclease (15 min at RT). The digested products were resolved on 18% denaturing PAGE. …

Electrophysiology assays:

LF N-terminal domain (LF_N) and PA were expressed and purified as described (21). To form heptameric PA oligomers, PA was nicked with trypsin (1:1000 w/w) at room temperature in buffer (20 mM TrisCl, pH 8) …

• Product #174 – Anthrax Protective Antigen, Activated (PA 63) from B. anthracis
• Product #171 – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

Reagents:

PA₆₃ was purchased from List Biological Laboratories, Inc., (Campbell, CA, USA). …

Channel Reconstitution into Planar Lipid Bilayers:

To form solvent-free planar lipid bilayers with the lipid monolayer opposition technique [81], we used …Single channels were formed by adding 0.5 to 1 L of 20 gmL¹ solution of PA₆₃ to the 1.5 mL aqueous phase in the cis-half of the bilayer chamber. Under this protocol, PA₆₃ channel insertions were always directional, as judged by channel conductance asymmetry in the applied transmembrane voltage. …

• Product #174 – Anthrax Protective Antigen, Activated (PA 63) from B. anthracis

Lethal Toxin, consisting of protective antigen and lethal factor were from List Biological Laboratories. …

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

Chemicals and Reagents:

All bacterial toxins were purchased from List Biological Laboratories (Campbell, CA, USA). …

LF FRET Drug Screen and Data Analysis:

For screening JHCCL drugs in 96-well plates, the reaction volume was 250l per well, containing 20mM HEPES pH 7.2, 5M MAPKKide conjugated with DABCYL and FITC (List Biological Laboratories, Inc), and 33M of JHCCL compound. Reactions were initiated by adding anthrax LF…

Cell Culture, Toxins Treatments, and Cell Viability Assays:

RAW264.7 mouse macrophage cells were maintained in DMEM (Invitrogen) supplemented with 10% FBS (Corning) and 100g/ml penicillin and streptomycin. …Cells were treated with drug hits for 1hour at 37C 5% CO₂. RAW264.7 cells were challenged with anthrax toxins that include LF and native 83kDa PA (for 3hours), LF and PA in the 63kDa form (for 6hours), P. aeruginosa exotoxin A (for 12hours), or Cholera toxin (for 12hours), which were also pre-treated with identical drug concentrations, such that the final toxins concentrations were 0.5, 0.5, 2.0, and 4.0g/ml respectively. …

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

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis
• Product #174 – Anthrax Protective Antigen, Activated (PA 63) from B. anthracis
• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

Toxin Neutralization Assay (TNA):

… Goat sera (starting dilution; 1:50) were doubly diluted in culture medium containing PA and LF (List Biological Laboratories, Inc …

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

Chemicals and reagents:

All bacterial toxins were purchased from List Biological Laboratories (Campbell, CA). …

Human B-lymphocytes sensitivity to Pseudomonas toxin-mediated lethality:

Human B lymphocytes were treated with serial dilutions of P. aeruginosa exotoxin A for 48hours.

Cellular drug screens:

RAW264.7 cells (10,000 per well) were seeded in 96-well plates 24hours before the assay. Cells were treated with compounds for 1hour, and then challenged with either 2g/ml PE or 4g/ml cholera toxin for 12hours. As rodent cells are insensitive to diphtheria toxin, C32 cells were treated with 2g/ml of diphtheria toxins for 24hours. …

Toxins treatments and cell viability assays:

Cells (10,000 per well) were seeded in 96-well plates 24hours before the assay. Cells were treated with Bithionol for 1hour. RAW264.7 cells were challenged with anthrax toxins that include LF or FP59 and PA83 or PA63 (for 6hours), PE (for 12hours), or cholera toxin (for 12hours) at 0.5, 2, and 4g/ml respectively. C32 cells were treated with 2g/ml of diphtheria toxin for 24hours. Determination of cells viability was performed by MTT assay….

MAPKK2 cleavage assay:

… Following pre-treatment, the cells were exposed to 0.5g/ml of PA and LF at 37C for up to three hours in the presence of 33M of Bithionol. …

Cellular cathepsin B and caspase-1 activity assays:

…Caspase-1 activities were induced by 1hour pretreatment of cells with 0.5g/ml of LF+PA, and was compared to cells un-induced by the toxin. …

BoNT/A light chain FRET assay:

The reaction volume was 250l per well in 96 wells plate, containing 50mM HEPES pH 7.4 containing 0.05% TWEEN 20, 5M SNAPtide (BoNT/A substrate peptide) conjugated with DABCYL and FITC (List Biological Laboratories, Inc), and 33M of Bithionol. …

• Product #160 – Exotoxin A from Pseudomonas aeruginosa
• Product #150 – Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae
• 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
• Product #174 – Anthrax Protective Antigen, Activated (PA 63) from B. anthracis
• Product #521 – SNAPtide Peptide Substrate (FITC/DABCYL) for C. botulinum Type A Neurotoxin
• Author did not specify which Cholera toxin was utilized.  List Labs provides Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae and Product #101 – Cholera Toxin from Vibrio cholerae.

…Recombinant protective antigen (rPA) was purchased from List Laboratories. …

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