Protein chips and DNA microarrays are manufactured in a similar fashion. Both involve spotting a biological component (such as DNA or proteins) onto a coated glass slide or other substrate such as gel pads and microwells. For protein chips, the slide must be coated with a substance that will bind the proteins without denaturing them. There are variety of binding methods available including absorption, cross-linking and hybridization.
Glass Slide Chips
Glass slide chips are advantageous because they can be used with standard microarray equipment and are inexpensive. They are prone, however, to evaporation of samples and cross contamination. The first methods for creating protein chips on glass slides involved placing the proteins in small gel pockets that were attached to the glass surface. The process then evolved to attaching the proteins with a crosslinking agent that coated the glass. This agent would bind to the primary amines on the proteins, while a 40% glycerol solution would prevent dehydration due to evaporation. Most array spotting is now done in a high humidity enviornment to control evaporation.
Using wells instead of coated glass slides reduces the problem of evaporation and cross contamination between spots. However, this method is more expensive and is not compatible with DNA microarray spotting equipment.
3D Matrix Gels
A third method is to use a 3D matrix created by polyacrylamide gel pads. This method limits evaporation and allows the proteins to remain in an aqueous environment. Additionally, this method allows for stronger binding of the proteins. Like the microwell method, this method is more expensive and can't be used with traditional DNA microarray spotting equipment.
Protein Attachment Methods
The surface of the chip must be modified to allow the proteins to bind properly. An attachment layer which usually consists of a sugary gel such as dextran-based hydrogel is coated onto the chip. The noise level for these types of attachment layers is high, however, because of the non-specificity of the medium. Cross-linkers can be used that covalently bind to certain groups on proteins, allowing for much more specific binding. The cross-linkers could, however, effect the conformation or activity of the proteins. It is important to remember that unlike nucleic acids, different proteins will be effected in different ways by the surface chemistry of the chip, i.e. a certain treatment will tightly bind some proteins but may denature others.
Protein Delivery Methods
Many protein chips made currently are spotted using automated technology, with spot densities greater than 30,000 spots per chip, though some low density chips may still be hand spotted. In addition to spotting, peptides can be synthesized on the chip via photolithography.
Biacore - makers of SPR-based protein interaction analysis systems
Bio-Rad Laboratories - makers of the ProteOn XPR36 Protein Interaction Array System
Ciphergen - makers of the ProteinChip® System Series 4000
EMD - makers of the ProteoPlex 16-Well Human Cytokine Array
Invitrogen - makers of ProtoArrayTM
LC Sciences - makers of custom peptide microarrays for kinase profiling, epitope binding and other applications
Plexigen - makers of the geneCubeTM
SCHOTT - Manufacturers of NexterionTM coated slides for protein microarrays
Sigma-Aldrich - makers of the PanoramaTM Ab Microarray Cell Signaling Kit
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