Saporin belongs to the well-characterized family of ribosome-inactivating proteins and serves as a control

Targeted SAP conjugates are powerful and specific lesioning agents used in the technique known as Molecular Surgery.

The ribosome-inactivating protein, saporin (from the seeds of the plant, Saponaria officinalis) is bound to a targeting agent (anything that is recognized on the cell surface and internalized).

The targeted conjugate is administered to cells (in vitro or in vivo). The targeting agent seeks out and binds to its target on the cell surface.

The conjugate is internalized, saporin breaks away from the targeting agent, and inactivates the ribosomes which causes protein inhibition and, ultimately, cell death.

Cells that do not have the cell surface marker are not affected.

Products

Products

ZAP conjugates are non-targeted saporin conjugates that “piggyback” on to your primary targeting agent (biotinylated material or antibody).

Biotin-Z Internalization Kit (for in vitro or in vivo use)

These kits convert biotinylated materials into targeted toxins and will allow you to evaluate the ability of your reagent to internalize upon binding to its receptor.

Streptavidin-ZAP (Cat. #IT-27) converts biotinylated materials into targeted toxins.

FITC-Streptavidin-ZAP (Cat. #IT-85) eliminate cells recognized by YOUR biotinylated targeting agent, FITC-labeled

MonoBiotin-ZAP (Cat. #BT-ZAP) eliminate cells recognized by YOUR streptavidinylated targeting agent

ZAP Antibody Internalization Kit (for in vitro use)

The Antibody Z-kit contains a Secondary Antibody conjugated to Saporin (ZAP), Control Conjugate, plus all the components needed to screen your primary antibody for internalization.

made with bivalent antibodies that recognize the whole IgG

made with monovalent (Fab) antibodies that recognize the whole IgG, without bivalent capping

made with monovalent (Fab) antibodies that recognize Fc region only

pHast Conjugates are one of our pHastest tools for quantitative testing of your primary antibody’s specificity, binding, and internalization, providing results in 1 day.

Fluorescence intensity increases over time, indicating internalization of the pHast conjugate. Data generated using an IncuCyte SX5.

How It Works

The pHast conjugate binds to your primary antibody via a secondary antibody cross-linked to a pH-dependent fluorescent reporter. This fluorescent reporter will increase intensity as the pH of its surroundings becomes more acidic, as evident when exposed to the environment inside a cell. pHast conjugates are fluorescent plate reader compatible and can be used to illuminate your lead antibody candidates in a 96-well plate format.

Also Available:

Streptavidin-pHast (PH-03)
Streptavidin-pHast binds to your biotinylated protein and contains a pH-dependent fluorescent reporter (pHast).

Target	Recommended Products made with whole IgG	made with monovalent (Fab)	made with monovalent Fc region (FabFc)
alpaca/llama IgG (VHH domain)	Alpaca-pHast (PH-05)
chicken IgY	Chick-pHast (PH-06)
goat IgG	Goat-pHast (PH-07)
guinea pig IgG	gPIG-pHast (PH-08)
human IgG	Hum-pHast (PH-09)	Fab-pHast human (PH-01)	FabFc-pHast human (PH-10)
human IgM	Hug-M-pHast (PH-11)		FabFc-pHast Hug-M (PH-04)
mouse IgG	Mab-pHast (PH-12)	Fab-pHast mouse (PH-02)	FabFc-pHast mouse (PH-13)
mouse IgM	Anti-M-pHast (PH-14)
rabbit IgG	Rab-pHast (PH-15)	Fab-pHast rabbit (PH-16)
rat IgG	Rat-pHast (PH-17)	Fab-pHast rat (PH-18)	FabFc-pHast rat (PH-19)
	made with bivalent antibodies that recognize the whole IgG	made with monovalent (Fab) antibodies that recognize the whole IgG, without bivalent capping	made with monovalent (Fab) antibodies that recognize Fc region only

Products

Products

Controls are a vital part of the scientific procedure; without them it is difficult to isolate the specific effects from the non-specific or artifactual. These control conjugates are the same molecular weight, consist of similar, comparable materials and are synthesized with the same protocols as the targeted conjugates. The difference is the cell-specific targeting agents are replaced with “blanks,” antibodies or peptides that have no specificity, and no ability to target cells.

ATS is pleased to announce Beta-release of a wide array of targeted toxins for use in eliminating specific cell types. This Beta-Testing Program will make new conjugates available to our customers sooner.

Each of the Beta products has had:

1. Saporin activity confirmed,
2. Peptide sequences published/confirmed, and/or
3. Antibody binding specificity published/confirmed.

Beta Products now available as catalog items:

Anti-CD105-SAP (Cat. #IT-80) previously BETA-015

Anti-CD44-SAP (Cat. #IT-72) previously BETA-004

Anti-PD-L1-SAP (Cat. #IT-45) previously BETA-014

Chlorotoxin-SAP (Cat. #IT-86) previously BETA-010

Ex4-SAP (GLP-1-SAP) (Cat. #IT-90) previously BETA-009

MOA-SAP (Cat. #IT-92) previously BETA-013

Orexin-B-SAP (Cat. #IT-20) previously BETA-031

PACAP-SAP (Cat. #IT-84) previously BETA-029

By Type

Custom Conjugates are powerful tools that specifically destroy targeted cell types. This technique has important research applications in determining the function or contribution of the targeted cells in a biological system or for the removal of interfering cell types.

Custom Conjugates are powerful tools that specifically destroy targeted cell types. This technique has important research applications in determining the function or contribution of the targeted cells in a biological system or for the removal of interfering cell types. Through the use of custom conjugates, you can eliminate any cell type.

Requirements for Successful Targeting

• A cell surface marker.
• A molecule, such as an antibody or a ligand, that binds to that marker.

Your targeting agent is conjugated to Saporin, a highly active and potent ribosome-inactivating protein.

Getting Started

• Select your targeting agent.
  As an example, for the specific and complete removal of mature T lymphocytes, use your own anti-transferrin receptor antibody; or obtain one from the ATCC.
• Use the appropriate Secondary Conjugate to screen your targeting agent for specificity and internalization.
• Contact us for a quote.
• Prepare for stunning, powerful research results!

Examples of Uses

Examine the role of targeted neurons in behavior, in gene expression and to determine the effect of these neurons on other neurons in the brain.

Selective removal of subtypes of the hematopoietic system, which has well-characterized cell surface markers.

Research Applications

Custom conjugates are valuable tools with the potential for applications in many areas.

In vitro, custom conjugates can be used to selectively eliminate cells expressing a specific receptor/antigen. This could be very useful in various cloning and gene trapping strategies where the goal is to select for cells based on expression (or suppression of expression) of a particular gene.

In vivo, custom conjugates can be used to specifically eliminate selected types of neurons for studies of the function of those neurons and responsiveness of animals to a variety of manipulations, including:

• Behavioral studies
• Drug treatments
• Physiological studies

Screening Targeting Agents

Use a Secondary Conjugate to test your antibody as a targeting reagent.

Proteins come in all shapes and sizes and don’t always contain a ready-to-conjugate binding site. This and other variables can cause you to look toward one-step kits, often leaving you with un-purified and un-verified conjugate.

Let the experts in the field help provide you with the most effective conjugate while retaining the functionality of each of the components.

Contact us now to get started discussing the strategy that best meets your needs.

Why biotinylate through Advanced Targeting Systems?

What you get with a “One-Step-Kit”

• Un-purified conjugate
• Un-verified conjugate
• Estimated concentration
• Reacts only with amine groups

VS

ATS Custom Biotinylation

• Discuss options available for your protein
• Removal of unbound biotin & protein
• Calculation of ratio of moles biotin/moles protein
• Concentration verified by BCA*
• Zero hands-on time for you

*Dependent upon peptide, but typically available for most proteins such as antibodies.

Leonardo Ancheta,
Vice President and Director of Custom Conjugations at ATS, talks about important things to watch for to avoid compromising your next experiment.

Companion Product

Biotin-Z Internalization Kit

The streptavidin-biotin interaction has found extensive use as a research tool. The bond created between streptavidin and biotin is rapid and essentially non-reversible, unaffected by most extremes of pH, organic solvents, and denaturing reagents. A variety of molecules including lectins, proteins, and antibodies, can be biotinylated and reacted with streptavidin-labeled probes or other detection reagents for use in biological assays.

It’s easy to test your biotinylated targeting agent using the Biotin-Z Internalization Kit. Each kit contains all the components needed to allow your targeting agent to be screened quickly and economically to provide specificity, functional binding, internalization, and EC50 determination.

The key component of the Biotin-Z Internalization Kit is Streptavidin-ZAP; Streptavidin attached to Saporin, the most potent (and safest for use in the laboratory) of the plant ribosome-inactivating proteins. Streptavidin-ZAP “piggybacks” onto YOUR biotinylated material in order to evaluate the ability of the reagent to internalize upon binding to its receptor. Using Streptavidin-ZAP and biotinylated targeting agents, specific cytotoxins can be created JUST BY MIXING!

You can also test your biotinylated targeting agent in vivo. Streptavidin-ZAP can be mixed with your biotinylated targeting agent and delivered specifically to eliminate cells that recognize your targeting agent.

Your scientific applications demand a purified and verified conjugation.
The experts at ATS can make your next conjugation a solid success with “zero” hands-on time for you.

Data sheets accompany all biotinylations and include:

• Protein Concentration Verified by Bicinchoninic Acid Assay (BCA assay)
• Average chemical ratio of moles of biotin to moles of protein
• Average effective molecular weight

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).