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June 19, 2019
Biochemistry
Farcasanu, M;Wang, AG;Uchaski, T;Bailey, LJ;Yue, J;Chen, Z;Wu, X;Kossiakoff, A;Tang, WJ;
Product: Anthrax Protective Antigen (PA), Recombinant from B. anthracis
… The column was washed with T205N100P0.1 followed by T205N100P0.1 plus 20 mM imidazole and eluted with T205N100P0.1 with 150 mM imidazole. Peak fractions were pooled and diluted tenfold with T20P0.1, loaded onto a Source Q anion exchange column, and eluted by a 0-1M NaCl gradient. Purified EF was concentrated to approximately 20 mg/mL and frozen at -80C. Protein quantitation was performed using extinction coefficients calculated from the known primary sequence of each protein on the Expasy ProtParam webserver. PA was purchased from List Labs. …
June 17, 2019
POSTER
Suryadi, K.; Shine, N.
Product: Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence
Materials and Methods
Anthrax lethal factor (Product #172 or #169), are products of List Biological Laboratories, Inc. The 96-well, black, flat bottom, non binding plates used for the fluorescent plate assay were from Corning (cat # 3991). Bovine plasma (cat # 7310806) was
purchased from Lampire Biological Laboratories.
Sample Preparation: Stock solutions of the fluorogenic substrates, MAPKKide® Plus (± biotin), were made 2.5 mM in
DMSO based on the peptide content determined by elemental analysis. The substrates were diluted in assay buffer: 20 mM
HEPES, pH 8.0 containing 0.1% Tween-20.
LF Activity Assays:
A rapid microplate assay method was evaluated for two ranges of LF: 10 to 1000 pg LF/ml 1:10 diluted bovine plasma and 5 to 250 ng/ml 1:5 diluted bovine plasma. Dilution of the bovine plasma was necessary in order to minimize background.
For the method detecting higher levels of LF (5 to 250 ng/ml 1:5 diluted bovine plasma), 10 μM MAPKKide Plus was added directly to the 1:5 diluted bovine plasma, and the assays were run at 37°C using the kinetic mode of the plate reader with
readings at 1 or 3 minute intervals. The samples were run in triplicate with 9 replicate blanks. The limit of detection was
calculated from the normal distribution of the blank samples (mean + 3 stdev; n = 9).
For the range 10 and 1000 pg/ml 1:10 diluted plasma, 1.25 μM MAPKKide Plus was added directly to the diluted bovine
plasma and the time-dependent increase in fluorescence was monitored at 37°C hourly for 5 hours. For the blank samples
containing no LF there were 3 sets of quadruplicates and the standard deviation was calculated from these three sets. At
each time point, the plate was read 5 times to increase the precision of the fluorescence readings. The standard curve was
analyzed using a linear regression fit forcing the intercept through the mean value of the blanks. The limit of detection was
calculated from the normal distribution of the blank samples (mean + 3 stdev; n = 3 sets of quadruplicates) and calculated as
pg LF/ml plasma using the standard curve. Subsequently, 6 data sets were evaluated, 2 data sets per day for 3 consecutive
days. The data is presented as the average of these 6 data sets, each with 4 replicate samples and 12 replicate blanks. At
each time point, the plate was read 3 times to increase the precision of the fluorescence readings.
• Product #169L – Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence
• Prodcut #172 – Anthrax Lethal Factor (LF), Recombinant from B. anthracis
• Product #532 – MAPKKide® Plus (AMC) Specific Substrate for Anthrax Lethal Factor
April 23, 2019
Elife
Li, Y;Li, KX;Hu, WL;Ojcius, DM;Fang, JQ;Li, SJ;Lin, X;Yan, J;
Product: Anthrax Edema Factor (EF), Recombinant from B. anthracis
Lep-vesicle-recycling endosome-exocyst complex inhibition tests:
The cell monolayers were treated with 35 nM edema factor or lethal factor plus 70 nM protective antigen (EF + PA or LF + PA) of anthrax toxin (List Biological Laboratories, USA), the inhibitor (EF + PA) of Rab11 and inhibitor (LF + PA) of Sec15 to block Rab11-Sec15 binding, for 24 hr at 37C (Guichard et al., 2010), and then infected with L. interrogans strain Lai at MOI100 for 8 hr. The subsequent steps and detection of Lep-vesicle-Rab11-Sec15/Sec3 co-localization were the same as above. …
April 11, 2019
Cell Death & Disease
Loomis, WP;den Hartigh, AB;Cookson, BT;Fink, SL;
Product: Anthrax Lethal Factor (LF), Recombinant from B. anthracis
Lactate dehydrogenase (LDH) assay:
Macrophages were pretreated for 30min with glycine or one of the other small-molecule inhibitors at the indicated dose. All chemicals were purchased from Sigma-Aldrich, with the exception of propofol (EMD Millipore). Late-log cultures of Salmonella typhimurium SL1344 grown in L-broth containing 0.3M NaCl were used to infect macrophages (MOI 10:1) for 90min, unless otherwise indicated. Cells were treated with anthrax lethal toxin comprised of 1g/ml protective antigen and 1g/ml lethal factor (List Biological) for 2h14. Cells were treated with the detergent-based lysis buffer included in the Cytotox 96 Kit (Promega) for 30min. Release of cytoplasmic LDH was determined from triplicate samples using the Cytotox 96 Kit and calculated as 100 (experimental LDHspontaneous LDH)/(maximum LDHspontaneous LDH).
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 #169L (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).
March 26, 2019
Analytical And Bioanalytical Chemistry
Lins, RC;Boyer, AE;Kuklenyik, Z;Woolfitt, AR;Goldstein, J;Hoffmaster, AR;Gallegos-Candela, M;Leysath, CE;Chen, Z;Brumlow, JO;Quinn, CP;Bagarozzi, DA;Leppla, SH;Barr, JR;
Product: Anthrax Protective Antigen, Activated (PA 63) from B. anthracis
… Recombinant EF was produced as previously described. Activated recombinant PA (PA63) was from List Biological Laboratories (Campbell, CA). …
March 25, 2019
The Analyst
Solano, MI;Woolfitt, AR;Boyer, AE;Lins, RC;Isbell, K;Gallegos-Candela, M;Moura, H;Pierce, CL;Barr, JR;
Product: Anthrax Lethal Factor (LF), Recombinant from B. anthracis
Experimental procedures –
Materials:
Recombinant anthrax toxins PA83, PA63, LF and EF were 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 #169L (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).
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).
March 7, 2019
International Journal Of Molecular Sciences
Pan, Z;Dumas, EK;Lawrence, C;Pate, L;Longobardi, S;Wang, X;James, JA;Kovats, S;Farris, AD;
Product: Anthrax Edema Factor (EF), Recombinant from B. anthracis
Efferocytosis Assay:
Efferocytosis was assessed by flow cytometry [68]. Briefly, 5 105/well monocyte-derived human M2(IL-4+IL-10+Dex) macrophages or M2(Dex) macrophages were preincubated with 10 nM recombinant PA (List Biological Laboratories, Campbell, CA, USA), 10 nM recombinant EF (List Biological Laboratories), or various concentrations of PA + EF (ET) for 4 h at 37 C in 5% CO2 atmosphere. Fluorescently-labeled apoptotic human PMN were preincubated with 10% serum, Protein S, or MFGE8 for 1 h unless otherwise noted and then added to the macrophages, resulting in a macrophage:PMN ratio of 1:5. After 1 h at 37 C in 5% CO2 atmosphere, the percentage macrophages containing intracellular (eFluor 670+) but not surface-bound (surface CD66b) PMN was assessed by flow cytometry.
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).
February 22, 2019
Vaccine
Lu, F;Mosley, YC;Carmichael, B;Brown, DD;HogenEsch, H;
Product: Anthrax Protective Antigen (PA), Recombinant from B. anthracis
… Proteins included … and recombinant protective antigen (rPA; List Biologicals, Campbell, CA). …
February 20, 2019
The EMBO Journal
Ren, G;Zhang, X;Xiao, Y;Zhang, W;Wang, Y;Ma, W;Wang, X;Song, P;Lai, L;Chen, H;Zhan, Y;Zhang, J;Yu, M;Ge, C;Li, C;Yin, R;Yang, X;
Product: Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence
… Activation of the NLRP1 inflammasome was achieved by treatment for 16 h with anthrax lethal toxin (2.5 lg/ml Anthrax Protective Antigen and 1 lg/ml Anthrax Lethal Factor mix, List Biological Laboratories, 171D and 169A). …
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 #169L (Anthrax Lethal Factor (LF-A), Recombinant from B. anthracis Native Sequence).
February 8, 2019
NPJ Vaccines
Weir, GM;MacDonald, LD;Rajagopalan, R;Sivko, GS;Valderas, MW;Rayner, J;Berger, BJ;Sammatur, L;Stanford, MM;
Product: Anthrax Protective Antigen (PA), Recombinant from B. anthracis
Vaccine preparation and immunization:
DPX-rPA and alum-rPA were provided by IMV Inc. (Halifax, Canada), but AVA was provided by the CDC. Test and vehicle/control article formulations were prepared fresh on the day of dosing in accordance with the method provided. DPX-rPA was prepared as previously described19 containing a poly I:C (Thermo Fisher) or Pam3CSK4 (EMC Microcollections) adjuvant and a 10:1 mixture of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine):cholesterol (Lipoid GmBH, Germany) or S100:cholesterol (Lipoid GmBH), see Supplementary Table 1 for complete formulation details for each figure. The source of rPA was DRDC, List Biologicals, or Pfenex Inc.26 …