Merck, R4658-5MG, PEPTITE-2000®

Application
INSTRUCTIONS FOR USE:
Use these procedures as a guideline to determine the optimal coating conditions for the culture system of coice. To maintain sterility, perform all operations in a laminar flow hood. Two options are provided:

Procedure A
1. Remove cap and add 5 ml of serum-free medium or PBS to the bottle.
2. Replace cap and vortex contents vigorously. Ensure that the PEPTITE-2000 is completely solubilized. The solution will remain slightly hazy.
3. Transfer desired volume of solution from the bottle to a dilution vessel. Dilute to desired concentration using serum-free medium or PBS. A typical working concentration may range from 0.1 to 10 μg/ml.
4. Add appropriate amount of diluted material to culture surface.
5. Incubate at room temperature or 37 °C, covered, for 1-2 hours.
6. After incubation, aspirate remaining material.
7. Rinse plates carefully with water and avoid scratching bottom surface of plates.
8. Plates are ready for use. They may also be stored at 2-8 °C damp or air dried if sterility is maintained.
9. Store remaining solubilized PEPTITE-2000 at 2 to 10 °C.
Additional note: Include divalent cations (Calcium, Magnesium, or Manganese) in cell attachment solution to obtain optimum cell binding.

Procedure B
1. Remove cap and add 5 ml of sterile 70% Ethanol
2. Replace cap and vortex contents. Ensure that the PEPTITE-2000 is completely solubilized.
3. Transfer desired volume of solution from the bottle to a dilution vessel. Dilute to the desired concentration using 70% Ethanol. Concentrations from 0.1 to 10 μg/ml should be tested.
4. Add appropriate amount of diluted material to culture surface.
5. Leave the coated container, uncovered, in a laminar flow hood until the wells are dry.
6. Rinse plates carefully with water and avoid scratching bottom surface of plates.
7. Plates are ready for use.
8. Store remaining solubilized PEPTITE-2000 at 2-10 °C.
PEPTITE-2000® is suitable to coat:
paramagnetic beads prior to incorporation with embryonic stem cells
Corning Transwell polycarbonate membrane inserts for promoting cell attachment
tosyl-activated magnetic beads in human colonic epithelial cells (HT-29) for binding experiments
gold nanorods

Features and Benefits
RGD PEPTITE-2000 is used to coat tissue culture plasticware for enhanced cell attachment and adhesion. The product has been specifically developed for ease-of-use for the researcher. It is provided in a 5 mg package size to provide sufficient quantity of product for coating a large surface area. The product has been sterilized and is ready-to-use after proper dilution. The optimal concentration for cell attachment and culture may differ for various cell types. Some experimentation will be required to determine the optimal conditions for individual cell culture systems. A typical working concentration may range from 1 to 100 μg/ml.
Analysis Note
Identity and purity qualitatively determined by SDS-PAGE
Other Notes
Ac-Gly-D-Arg-Gly-Asp-Ile-Pro-Ala-Ser-Ser-Lys-Gly-Gly-Gly-Gly-Ser-D-Arg-Leu-Leu-Leu-Leu-Leu-Leu-D-Arg-NH2
The tripeptide Arg-Gly-Asp (RGD) is an important protein sequence in the binding of proteins to cell surfaces. The RGD motif was initially identified in fibronectin as the minimal sequence that mediates cell attachment. Research has also shown that the RGD motif in present in other proteins such as vitronectin, osteopontin, collagens, thrombospondin, fibrinogen and many other factors. Many adhesive cell surface receptors recognize the RGD sequence for cell attachment.
Legal Information
PEPTITE-2000 is a registered trademark of Advanced BioMatrix, Inc.