Adhesion ligands on inert background back to list

See text on Nano-clustered adhesive ligand on inert backgrounds below since these substrates are basically the same but present a uniform ligand density.

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Inert (non adhesive) surfaces back to list

These substrates inhibit adhesion of proteins and cells, although some non-specific adhesion of peptides or other small molecules may be observed.

Lead time: 3-10 days for preparation. Storage: Dessicated. Prefer that they be used right away.

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Nano-clustered adhesion ligands on inert background back to list

These substrates are comb-copolymers with ~50 nm islands containing 1-8 peptide adhesion ligands per cluster, which resist adsorption of cell-secreted matrix molecules. They are stable for at least a week in culture. Substrates are all made-to-order and thus in principle the user can provide specific peptides. We have prepared mixed substrates containing two different peptides that have only minor differences in sequence (substitution of RGE for RGD within a linear sequence). Peptides that have been attached to the surfaces and shown to be active for cell adhesion or other behaviors are listed below;

Lead time: Coverslips with linear RGD-containing sequences are generally available in 2-3 weeks. Preparation of other substrates depends on availability of the peptide.

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Tethered EGF on adhesive background back to list

These substrates present murine EGF by tethered by the amino terminus on a 2 nm tether at a density of >400 EGF per micron squared. Substrates should be coated with adhesion molecules to engender cell adhesion.

Lead time: variable, contact Linda Griffith

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Cell migration gradients back to list

Ligand surface composition gradients are formed by coupling dynamically controllable electrochemical potential gradients with electrosorption reactions of organothiols to vary the composition profile of one- or two-component self-assembled monolayers (SAMs) laterally and then immobilizing the desired ligand on the SAM gradient surface. The advantage of the electrochemical approach is that the position and width of the gradients are readily controllable in both space and time, even after initial formation. These ligand gradients are formed on 20-50nm thick gold films on glass (light transmission). We can provide glass slide with thicknesses of 0.1 and 0.4 mm (for short working distance objective). Gold films thicker than 50nm present light transmission difficulties, although fluorescence may still be probed by epi-illumination. The gradient overall length is flexible from 1mm to 5cm. Within the CMC, the FN gradient has been used for cell migration assays in the Schwartz and Haugh labs.

Lead time: 3-7 days for preparation. Storage: 4 C in PBS buffer.

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Cell adhesion peptide and protein monolayer gradient back to list

RGD-containing peptide organomercaptans are formed by reacting a peptide containing the RGD sequence with MUA. By applying an electrochemical potential gradient on RGD-thiol SAMs, RGD peptide can be stripped from regions of the surface where the local potential favors desorption. The bare regions can then be back-filled with protein adsorption-resistant MUD (mercaptoundecanol), OT (octanethiol) or PEG (ethylene glycol oligomer), creating a two-component full monolayer.

The reactive thiol MUA is merely illustrative. It can react with free amine groups on protein (Fn) by using activating agents N-hydroxysuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Backfilling with protein resistant agents (PEG, MUD, OT), produces a two-component gradient. Adjusting MUA concentration can control Fn coverage. The gradient slope and position can be easily tuned by changing the applied potential window ( V). Gradient slope range is typical. Gradient slopes within the range, e.g. 0-30% in the Fn/PEG system, are also possible. Substrates with defined surface coverage are special cases, or "zero-slope" gradients. Because the spatial rate of change is controlled electrochemically, it is possible to specify quantitatively the desired gradient slope. For example, 0.5% - 1.0% Fn over 2 mm. The UIUC group is happy to consult with experimental groups working in signaling and motility about their needs (contact us).

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Cell signaling molecule gradients back to list

Amino-terminated thiols, such as aminoundecanethiol (AUT) can be patterned into gradients and reacted with N-hydroxysuccinimidyl esters of the biomolecule of interest. This strategy is used almost exclusively with human epidermal growth factor (hEGF) gradients. hEGF coverage can be controlled by adjusting AUT coverage and AUT coating concentration. The gradient slope and position are also controllable. Other coupling strategies which have not been explicitly demonstrated with gradient structures, e.g. maleimide-cysteine coupling, biotinylated thiols-avidin-biotinylated protein, etc. should also work well.

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Multi-ligand gradients back to list

In order to control heterotypic receptor interactions, we are working on a multi-ligand gradients with adhesion proteins, e.g. Fn, and signaling molecules, e.g. EGF. Such interactions may be important in construction of physically stable bonds in focal adhesions or for facilitating signaling interactions. The coverage of these two ligands can be adjusted by changing their ratio in the immobilization solution. The gradient slope and position are also controllable.

All of these characterization tools are available at UIUC, and many are also available at other partner institutions. The UIUC group can collaborate in transferring expertise in gradient characterization in those cases where investigators wish to port the techniques to their own laboratories or provide sufficient characterization for fully competent exploitation of gradient structures. Determining what level of characterization is needed in any particular instance is best done by consultation. The UIUC group is also constantly looking to update and improve the surface biomolecular characterization tools for cell migration-relevant surfaces and is eager to collaborate on such projects.