CTb injections into the VTA:
The retrogradely transported marker CTb (List Biological Laboratories, Campbell, CA, USA) was unilaterally injected by iontophoresis into the VTA of 16 rats. CTb is considered to be a very sensitive retrograde tracer when visualized by immunohistochemistry. ... A hand drill was used to expose the brain surface above the VTA and a borosilicate glass micropipette (tip diameter 5–10 μm) filled with a low-salt CTb solution (0.5% made in 0.1 M phosphate buffer, pH 6.0) was lowered into the brain at the following stereotaxic coordinates: AP, −5.5 mm from bregma; L, −0.7 mm from the midline; and V −7.2 mm from the dural surface (Paxinos & Watson, 2005). CTb was delivered by applying positive pulses of 1–4 μA (7 s on, 7 s off) for 10 min through a chlorinated silver wire placed in the micropipette and connected to a current generator (Bionic Instruments, France). The micropipette was withdrawn 10 min after the CTb ejection, the scalp incision was sutured and rats were returned to their home cages for recovery.
Cholera toxin (CT) was purchased from List Biological Laboratories Inc. (Campbell, CA, USA).
Author did not specify which Cholera toxin was utilized. List Labs Product #101B/C - Cholera Toxin from Vibrio Cholerae has been discontinued; however, List Labs still provides Product #100B - Cholera Toxin (AZIDE-FREE) from Vibrio cholerae.
To follow, are our recommendations for transitioning from Product #101B/C to Product #100B:
Product #101B, Cholera Toxin from Vibrio cholerae (1 mg), has been discontinued. Product #100B, Cholera Toxin (AZIDE-FREE) from Vibrio cholerae, may be substituted for Product #101B, if the user accounts for the formulation differences. Both products contain the same quantity of toxin per vial (1.0 mg). For Product #100B, reconstitution with 1.0 mL of water results in a 1 mg/mL toxin solution in a buffer with the following component concentrations: 0.05 M Tris, 0.2 M NaCl, 0.001 M Na2EDTA at pH 7.5. For Product #101B, reconstitution with half that amount of water (0.5 mL) resulted in a 2 mg/mL toxin solution with the same concentrations of the buffer components, and in addition, Product #101B contained 0.003 M NaN3. The user may add the sodium azide preservative to product #100B, if desired.
Product #101C, Cholera Toxin from Vibrio cholerae (2 mg), has been discontinued. Product #100B, Cholera Toxin (AZIDE-FREE) from Vibrio cholerae, may be substituted for #101C, if the researcher accounts for the formulation differences. While Product #101C contained 2.0 mg per vial, Product #100 contains 1.0 mg per vial. When Product #100B is reconstituted with 1.0 mL of water, the resulting 1 mg/mL toxin solution will have buffer components with the following concentrations: 0.05 M Tris, 0.2 M NaCl, 0.001 M Na2EDTA at pH 7.5. For Product #101C, reconstitution with less than half that amount of water (0.4 mL) resulted in a 5 mg/mL toxin solution in the same buffer, and in addition, Product #101C contained 0.003 M NaN3. The user may add the sodium azide preservative to product #100B, if desired.
Large quantities of Product #101B or Product #101C may be obtained through Bulk Requests and Customer Orders.
The following FRET peptides have been designed at List Biological Laboratories as substrates for the botulinum toxin enzymes. SNAPtide®, Product #520 and #521 (U.S. Patent, No. 6,504,006 ) is readily recognized and cleaved by the Botulinum toxin type A (BTA), Product #130, and the BTA light chain (LcA), Product #610A. One of the substrates, Product #520, contains an oAbz/DNP FRET pair and the other, Product #521, a FITC/DABCYL FRET pair. Representative data are given below. VAMPtide®, Product #540 is readily recognized and cleaved by the Botulinum toxin type B light chain (LcB), Product #620A. This FRET substrate contains an oAbz/DNP FRET pair. SNAP Etide™, Product # 550 is readily recognized and cleaved by the Botulinum toxin type E light chain (LcE), Product #635A. This FRET substrate contains an oAbz/DNP FRET pair.
(LF) is the enzymatic component of anthrax lethal toxin which specifically cleaves the MAPK-kinase proteins. LF is also an ideal target for therapeutic inhibitors. FRET substrates for LF, MAPKKide®, Product #530 and 531, have been designed at List Biological Laboratories. One of the substrates, Product #530, contains an oAbz/DNP FRET pair and the other, Product #531, a FITC/DABCYL FRET pair. Product #531 is especially well suited for high throughput screening for the IC50 and inhibitor modality of potential inhibitors. ...
SNAP-EtideTMsubstrate (Product #550) and botulinum neurotoxin type E light chain, recombinant (Product #635A), are both products of List Biological Laboratories, Inc.
Continuous assays were performed on a SPECTRA max GEMINI XS fluorescence microplate reader (Molecular Devices, Sunnyvale, CA) using Greiner FLUO-TRAC black flat-bottomed plates (E&K Scientific, Campbell, CA). Stock solutions of the FRET substrate was made in dimethylsulfoxide (DMSO). Final dilutions were made in the appropriate buffer. Plates were equilibrated at 37°C for 15 min prior to initiation of the reaction. For all experiments the time-dependent increase in fluorescence intensity was monitored at 37°C. The excitation wavelength was set to 321 nm and emission to 418 nm.
FRET assays were performed to test the activity of LcE with SNAP-EtideTMas a function of pH, Tween-20 and ZnCl2. Three separate experiments were performed (Figure 1). The cleavage reaction was initiated with addition of 5 nM LcE to the wells containing 10 µM SNAP-EtideTM in the appropriate buffer. Initial velocities of cleavage in RFU/sec were evaluated and compared for each assay in order to determine the optimum buffer conditions for the reaction.
LcE titration experiment was performed in 50 mMHEPES, pH 7.8, 0.1% Tween-20, using 10 µM SNAP-EtideTM. LcE was prepared at 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, and 0.039 µM concentrations. Following equilibration, the cleavage reaction was initiated with addition of 10 µM SNAP-EtideTM. Initial velocities of cleavage were plotted against LcE concentration (Figure 2).
Dilutions of SNAP-EtideTMwere prepared in 50 mM HEPES, pH 7.8, 0.1% Tween-20 to achieve 70, 60, 50, 40, 30, 25, 15, 7.5, 3.75, 1.88, and 0.94 µM concentrations. The reaction was initiated with addition of 10 nM trypsin into each well. End point readings were taken after 50 min. A second round of 10 nM trypsin was added to each well in order to achieve total enzyme digestion. The maximum fluorescence reached was graphed as RFU/5000 against SNAP-EtideTM concentration (Figure 3A). An identical experiment was run using 2.5 nM LcE for digestion of SNAP-EtideTM. Initial velocities of cleavage were graphed in RFU/sec against substrate concentration (Figure 3B).
Inner Filter Effect Correction:
Dilutions of SNAP-EtideTM were prepared in 50 mM HEPES, pH 7.8, 0.1% Tween-20 to achieve concentrations ranging from 250 µM to 2 µM. Fluorescence end point readings of SNAP-EtideTM at each concentration were recorded. In order to determine theinner filter effect at each substrate concentration another set of end point fluorescence (RFU) readings were recorded after addition of 5.0 µM free o-Abz-Lys. Fluorescence intensity obtained for SNAP-EtideTM was then subtracted from the fluorescence intensity obtained for SNAP-EtideTMand o-Abz-Lysin order to obtain fluorescence for the free o-Abz-Lyspeptide. The decrease in fluorescence of the o-AbzLysin the presence of SNAP-EtideTM reflects the inner filter effect (Table 1). A correction factor is obtained for each SNAP-EtideTM concentration:
correction factor = RFU (o-Abz-Lys) at each [SNAP-EtideTM] RFU (o-Abz-Lys)
Initial reaction rates were obtained for each substrate concentration after addition of 2.5 nM LcE. The rates were corrected as given in Table 1. The plots of initial velocity versus SNAP-EtideTM concentration (Figure 4)indicates a decreasing rate of cleavage at concentrations of substrate greater then 100 µM. This is consistent with substrate inhibition. The kinetic data was analyzed using the substrate inhibition equation from Kaleida Graph software:
ax b+(x(1+x/c)) , where a = Vmax, b = Km, and c = Ki, competitive inhibition constant
Lethal Factor protease assay:
Lethal Factor (20 nM final concentration) and MAPKK substrate (MAPKKide® 12.5 µM, final concentration) were purchased from List Biological Laboratories, Campbell, CA and used according to the fluorescence resonance energy transfer (FRET) method. ...
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).