ATS is known as the “saporin company” and initially focused on providing research tools for the Neuroscience research community.
Over the years these tools have been used in all areas of scientific research and drug development.
The Company’s current product line includes targeted toxins, antibodies and custom services designed to assist scientists in the study of system functions, cell functionality, diseases and disorders.

Advanced Targeting Systems pioneered the use of targeted conjugates for use in a technique called “Molecular Surgery.”
These products include specific lesioning agents for cholinergic basal forebrain neurons, noradrenergic and adrenergic neurons, macrophages and microglia and a pan-neuronal agent that strikes an epitope expressed on all neurons.
In addition, ATS makes “secondary conjugates” which allow users to convert their own targeting agents into specific cytotoxic tools and to screen antibodies for internalisation.


Product Categories

Conjugate Protocols

CONCENTRATION CALCULATIONS
Convert molarity to mg/ml and mg/ml to molarity

Normally, our concentrations are given in units of milligrams per milliliter (mg/ml), but often data sheet quality assurance data are expressed in molarity (moles per liter, M) or fractions of that (e.g., micromolar (µM), 10-6 M, or nanomolar (nM), 10-9 M). Here is how to convert between these units.

FROM (mg/ml) TO molarity (M):

Divide the concentration (mg/ml) by the molecular weight. We will use the example of a typical immunotoxin that has a molecular weight of 210,000 grams per mole (or mg/mmole or kDa) (the molecular weight is usually found on the data sheet) and a common concentration is 1.0 mg/ml.

1.0 mg/ml


2.1 x 105 mg/mmole

 = 0.48 x 10-5 mmole/ml
= 4.8 x 10-6 mmole/ml

In the first line, the mg units cancel each other, leaving units of mmole/ml that is equal to moles/liter or molar (M). Therefore, 0.48 x 10-5 mmole/ml = 0.48 x 10-5 M or 4.8 x 10-6 M. This, of course, can be expressed as 4.8 µM, or 4.8 micromolar.

In summary: concentration (grams per liter) ÷ molecular weight (grams per mole) = moles per liter.

FROM molarity (M) TO (mg/ml):

Multiply the molar concentration (M, or moles per liter) by the molecular weight. In an example of an immunotoxin at 1.0 nM concentration (1.0 nmoles per liter or 1.0 x 10-9 M or 1.0 x 10-9 moles per liter) again using as an example, a targeted toxin of molecular weight 210,000 grams per mole (or mg/mmole or kDa):

1.0 x 10-9 moles per liter x 2.1 x 105 grams per mole  = 2.1 x 10-4 grams per liter
= 2.1 x 10-1 µg/ml
= 0.21 µg/ml

In summary: molar concentration (moles per liter) x molecular weight (grams per mole) = grams per liter.

Antibody Protocols

1. Coat maxisorp well plates (Nunc) with a solution of antigen (the dilutions for antigen range from  4-50 µg/ml, please see data sheet) in sodium carbonate buffer 0.05 M (pH 9.6) containing sodium metabisulfite (SMB) (Acros) 0.001 M, for sixteen hours at 4°C.

2. Saturate well plates with a solution of PBS (pH 7.3) containing 2.5 g/l of BSA (Acros), 0.05% Tween 20 (Acros) and SMB 0.001 M for one hour at 37°C.

3. Wash with PBS/0.5% Tween three times.

4. Dilute primary antibody as per antibody data sheet in PBS containing 2.5 g/l BSA, 10% of glycerol and SMB 0.001 M, 200 µl by well plate (incubating for 2 hours at 37°C).

5 . Wash with PBS/0.5% Tween three times.

6. 200 µl of peroxidase-labeled goat anti- “primary antibody host species” IgG (Sigma) diluted (1/10,000) in a solution of PBS containing 2.5 g/l BSA, 10% of glycerol, 0.5% Tween and SMB 0.001 M, will be applied by well plate (for one hour at 37°C).

7. Rinse well plates with PBS/0.5% Tween three times.

8. And finally, develop the peroxidase by incubating 200 µl by well plate of a citrate 0.1 M/phosphate, 0.2 M (pH 5) solution containing 0.4% of OPD (Sigma) and 0.03% of hydrogen peroxide (Acros) for ten minutes in the dark. After that, stop the reaction by the addition of 50 µl of 2 M HCl.

9. The optical density will be measured at 492 nm, to obtain the different values (IC50).

Perfusion protocol for Adult male Sprague Dawley (weight around 0.5 kg) :

1. The animals can be deeply anaesthetized, for example with urethane (0.5-1.5g/kg, intraperitoneal).

2. Heparinize and perfuse via the ascending aorta with 100 ml of cold physiologic saline (0.9% NaCl) and with the following fixative solution:

a) 300 ml of cold 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate-buffer (PB), pH 7.2 (two minutes).
b) 600 ml of cold 4% paraformaldehyde and 2% glutaraldehyde in 0.1 M PB, pH 7.2 (ten minutes).
c) Dissect out the brains and place in a solution of 4% paraformaldehyde in 0.1 M PB, pH 7.2, at 4°C for twelve to sixteen hours.
d) Before the brains are cut on a freezing microtome, infuse the brain in increasing concentrations of sucrose (a first bath of 5% of sucrose in PBS until the brains sink), after that repeat the same process in a solution with a higher level of sucrose (10%, 20%, 25% and finally 30%).

3. Cut serial sections around 50 µm-thick. Keep at 4°C in PBS (0.1 M, pH 7.2) and process for
immunostaining.

Immunostaining protocol:

1. In order to avoid possible interference with endogenous peroxidase, treat free-floating sections with distilled water containing NH3 (20%), H2O2 (30%) and NaOH (1%) for 20 minutes (other method is using a solution with 33% of H2O2 and 66% of methanol).

2. Wash the sections for 20 minutes in 0.15 M phosphate-buffered saline (PBS) (pH 7.2).

3. Pre-incubate for 30 minutes in PBS containing 10% of normal horse serum and 0.3% of Triton X-100 (mixed solution).

4. Incubate at room temperature (1 hour 30 minutes) and overnight at 4°C mixing with a solution containing primary antibodies.

5. Wash for 30 minutes with PBS.

6. Incubate for 60 minutes at room temperature with biotinylated anti-rabbit IgG (Vector) diluted 1/200 in PBS.

7. Wash for 30 minutes with PBS.

8. Incubate sections for 1 hour with a 1/100 diluted avidin-biotin-peroxidase complex (Vectastain).

9. Wash for 30 minutes with PBS.

10. Wash with Tris-HCl buffer (pH 7.6)(10 minutes).

11. Develop the tissue-bound peroxidase with H2O2 using 3, 3′ diaminobenzidine as chromogen.

12. Rinse the sections with PBS and coverslip with PBS/Glycerol (1/1).

Specific Product Protocols

AB-N34 Antibody to Choline Acetyltransferase (ChAT) Rabbit Polyclonal Immunostaining
AB-N39 Antibody to Melanopsin, affinity-purified Immunostaining
AB-T01 Anti-Conjugated Glutathione Rabbit Polyclonal ELISA
AB-T03 Anti-Conjugated 5-Hydroxytryptamine (Serotonin) Rabbit Polyclonal ELISA, Immunohistochemistry
AB-T06 Anti-Conjugated Noradrenaline Rabbit Polyclonal ELISA, Immunohistochemistry
AB-T07 Anti-Conjugated Dopamine Rabbit Polyclonal ELISA, Immunohistochemistry
AB-T08 Anti-Conjugated L-Glutamate Rabbit Polyclonal ELISA, Immunohistochemistry
AB-T10 Anti-Conjugated GABA (Gamma-Aminobutyric acid) Rabbit Polyclonal ELISA, Immunohistochemistry
AB-T11 Anti-Conjugated Dopamine Mouse Monoclonal ELISA, Immunohistochemistry
AB-T12 Anti-Conjugated L-Glutamate Mouse Monoclonal ELISA, Immunohistochemistry
AB-T16 Anti-Conjugated Histamine Mouse Monoclonal ELISA
AB-T023 Anti-Conjugated Glycine Rabbit Polyclonal ELISA, Immunohistochemistry
AB-T029 Anti-Conjugated Acetylcholine Mouse Monoclonal ELISA, Immunohistochemistry
AB-T030 Anti-Conjugated Tyrosine Rabbit Polyclonal ELISA
AB-T044 Anti-Conjugated Trans-Hydroxyproline Rabbit Polyclonal Immunohistochemistry
AB-T048 Anti-Conjugated D-Serine Rabbit Polyclonal ELISA, Immunohistochemistry
AB-T067 Anti-Conjugated L-Dihydroxyphenylalanine (L-DOPA) Rabbit Polyclonal ELISA, Immunohistochemistry
AB-T070 Anti-Conjugated Octopamine Rabbit Polyclonal ELISA, Immunohistochemistry
AB-T071 Anti-Conjugated Tyramine Rabbit Polyclonal ELISA, Immunohistochemistry
AB-T079 Anti-Melatonin Rabbit Polyclonal ELISA
AB-T080 Anti-Conjugated 5-Methoxytryptophan Rabbit Polyclonal ELISA, Immunohistochemistry
AB-T086 Anti-Conjugated Myristic Acid Rat Polyclonal ELISA
AB-T125 Anti-Conjugated NO-L-Cysteine Mouse Monoclonal ELISA, Immunohistochemistry, Western Blot
AB-T129 Anti-Conjugated Indole 3 Acetic Acid Rabbit Polyclonal ELISA
AB-T147 Anti-Conjugated Folic acid Rat Polyclonal ELISA, Immunohistochemistry
AB-T168 Anti-Conjugated Uric Acid Rabbit Polyclonal ELISA, Western blot
AB-T171 Anti-Conjugated L.Kynurenine Antibody ELISA, immunocytochemistry, immunohistochemistry, immunoblotting

pHast Protocols

100 mcg, 250 mcg, 1000 mcg

a tool to test antibody specificity, binding, and internalization with results in one (1) day

Fab-pHast in conjunction with your antibody can be analyzed via  fluorescent microscope, fluorescent plate reader, or flow cytometry. In order to  establish the EC50 of your antibody a fluorescent plate reader is recommended. A sample protocol for determining internalization via Fluorescent Plate reader

pHast Ab Internalization Assay

Parental HEK-293 cells, and HEK-293 cells transfected with the p75 receptor, were plated in a  96-well plate overnight. Titrated 192-IgG antibody (Cat. #AB-N43) was incubated at RT with 50 nM of Fab-pHast Mouse (Cat. #PH-02) for 20 minutes prior to addition to cells. Plates were  incubated overnight to allow maximum internalization, but a few hours is sufficient for detection.

Plates were read on a Spectra Max Gemini EM (Ex: 532nm/Em: 560nm). Data analysis was done by PRISM (GraphPad, San Diego).

Fab-pHast Internalization Protocol

1. Determine the number of cells needed for the planned number of plates.  Cells are plated in the center 60 wells in 90 μl of media per well.

2. Plate cells in a 96-well black, clear bottom plate or all black plate. The clear  bottom plate allows visualization of antibody internalization using a microscope. Cells are usually plated at 20,000 cells per well.

3. Transfer 100 μl of media into the wells around the edge of a 96-well plate.  These wells simply offer some protection from evaporation for the experimental wells.

4. Incubate plates for 20-24 hours before treatment.

5. It’s recommended to use a constant concentration of Fab-pHast at 50 nM and titrate your antibody. A good starting concentration of antibody is 10 nM. Optimization of concentration and dilutions will need to be established. High concentrations of unconjugated antibody may act as an inhibiter of fluorescent activity.

6. Make a stock solution of 500 nM of Fab-pHast, 10X the concentration that will be used in each well.

7. Add your desired concentrations of antibody to the Fab-pHast, 10X the desired concentration, and incubate at room temperature for 20 minutes.

8. The first and last experimental columns (2 and 11 in most plates) of cells are controls, only medium or Fab-pHast alone is added to these wells.

9. Add 10 μl of your 10X concentrations of Fab-pHast with titrated antibody to each experimental well.

10. Mix the plate gently on a plate mixer for 1-2 min, then incubate overnight to allow internalization. (Internalization can start to be detected in 1 hour, but maximal fluorescence occurs typically 19 hours after addition of the Fab-pHast conjugated antibodies).

11. To achieve a higher sensitivity, replace media with PBS before reading the plate.

12. Read the plate in a fluorescent plate reader at Ex: 532 nm/Em: 560 nm.

13. The absorbance of the sample wells is compared to the control wells to establish a curve.

14. The EC50 of your antibody can be derived from the curve, using PRISM software (GraphPad, San Diego).

Streptavidin-pHast [PH-03]
100 mcg, 250 mcg, 1000 mcg

a tool to test biotinylated protein specificity, binding, and internalization with results in one (1) day

Streptavidin-pHast in conjunction with your biotinylated protein can be analyzed via fluorescent microscope, fluorescent plate reader, or flow cytometry. In order to establish the EC50 of your antibody a fluorescent plate reader is recommended.

A sample protocol for determining internalization via Fluorescent Plate reader

Streptavidin-pHast Internalization Protocol

1. Determine the number of cells needed for the planned number of plates. Cells are plated in the center 60 wells in 90 μl of media per well.

2. Plate cells in a 96-well black, clear bottom plate or all black plate. The clear bottom plate allows visualization of antibody internalization using a microscope. Cells are usually plated at 20,000 cells per well.

3. Transfer 100 μl of media into the wells around the edge of a 96-well plate. These wells simply offer some protection from evaporation for the experimental wells.

4. Incubate plates for 20-24 hours before treatment.

5. Mix an equimolar amount of Streptavidin-pHast to your biotinylated protein. Incubate at room temperature for 20 minutes. Optimization of concentration and dilutions will need to be established. High concentrations of unconjugated antibody may act as an inhibiter of fluorescent activity.

6. Serial dilute your desired concentrations of Streptavidin-pHast-Biotinylated-Protein in microcentrifuge tubes, 10X the desired concentration planned for the plate.

7. The first and last experimental columns (2 and 11 in most plates) of cells are controls, only medium or Streptavidin-pHast alone is added to these wells.

8. Add 10 μl of your 10X concentrations of Streptavidin-pHast-Biotinylated-Protein to each of your plates.

9. Mix the plate gently on a plate mixer for 1-2 min, then incubate overnight to allow internalization. (Internalization can start to be detected in 1 hour, but maximal fluorescence occurs typically 19 hours after addition of the Streptavidin-pHast conjugated proteins).

10. To achieve a higher sensitivity, replace media with PBS before reading the plate.

11. Read the plate in a fluorescent plate reader at Ex: 532 nm/Em: 560 nm.

12. The absorbance of the sample wells is compared to the control wells to establish a curve.

13. The EC50 of your antibody can be derived from the curve, using PRISM software
(GraphPad, San Diego).

Technology Tutorial

Advanced Targeting Systems’ technology – Molecular Neurosurgery – is a modification of one of the most widely used techniques in the neurosciences: lesioning of a region by surgical means and observation of the effect.


First for Antibodies

Stratech Scientific have been successful in a recent antibodies tender from the Southern Universities Purchasing Consortium. In the award criterion Read More…