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Protein Microarray Chips

Protein and Antibody Microarrays

Table of Contents

Introduction and Background to Protein and Antibody Microarrays

Types of Antibody and Protein Chips

Protein and Antibody Attachment Methods - Creation of a Microarray Chip

Protein Chip Delivery Methods

Protein Chip Capture Molecules and Their Limitations

Antibody Microarrays: Problems and Solutions

Protein Microarray Detection Methods and Analysis

Protein Production for Protein Arrays

Applications of Protein Arrays and Protein Chips

Protein Microarrays: Future Directions and Conclusions

References for Protein and Antibody Microarrays

Introduction and Background to Protein and Antibody Microarrays.

In spite of recent advancements in our understanding of molecular biology, in many cases we are unable to implicate specific proteins with a disease. Genomics and microarray technology have allowed us to analyze thousands of mRNAs at one time and determine whether mRNA expression is changed in disease states. However, researchers have long known that the concentration of an mRNA within a cell is poorly correlated with the actual abundance of that protein (1,2,3). This is due to the fact that the rate of degradation of individual mRNAs and proteins differ, post-transcriptional control of protein translation (4), a number of post-transcriptional modifications of protein (5), and protein degradation by proteolysis (6).
By measuring the amount of the specific protein directly, we are measuring a true level of gene function. However, when one takes into consideration the large number of post-translational modifications, human cells may contain a million or more different protein variants, any of which could be altered in disease making the task of analyzing all of them a huge task. Protein microarrays or protein chips may allow for a solution to this problem. A slide or "chip" could be spotted with thousands of known antibodies or peptides like a DNA microarray, a biological sample spread over the chip, and any binding determined. Binding could also be analyzed using standard proteomic techniques such as time-of-flight mass spectrometry (MS) and peptide mass fingerprinting. Protein chips can thus become a fast and high-throughput method of profiling protein changes in disease. (7)

Protein chips have the potential to function in many other applications including the study of protein–protein, protein–drug interactions, DNA-protein interactions, protein localization, antigen-antibody interactions, enzyme-substrate, and receptor-ligand interactions all of which may be amendable to array-type high-throughput screening (7,8).

Two approaches have been used in order to characterize multiple proteins in a biological sample. The first approach is 2-dimensional gel electrophoresis, which has been widely used to separate and visualize up to 2000-10,000 proteins in a single experiment by excision and identification by mass spectrometry (MS) (9).  This method is both time consuming and even with MS, only the most abundant proteins can be detected. Also, reproducibility is problematic, even though pre-cast gels and commonly used reagents, protocols, and hardware components have led to improved performance (17). Due to the limitations of 3D-gel separation technology, increasing attention is focusing on the development of the second approach, the development of protein microarrays as an alternative and complementary approach (10-12).
The theoretical background for protein microarray-based ligand binding assays was initially developed by Ekins et al. in the late 1980s (13-16). According to the model, antibody microarrays not only would permit simultaneous screening of an analyte panel, but would also be more sensitive and rapid than conventional screening methods. Interest in screening large protein sets only arose as a result of the achievements in genomics by DNA microarrays and the Human Genome Project (17).

The first array approaches attempted to miniaturize biochemical and immunobiological assays usually performed in 96-well microtiter plates (18-19). 96-well antibody arrays were first created with 144 elements each for standard enzyme-linked immunosorbent assays (ELISAs) (20). Similar arrays were used to measure prostate-specific antigen (PSA) and cytokines (21).

Filter membranes were also initially used because of their superior protein binding capacity. They were mostly probed with antibodies using ELISA techniques. A low density array of 48 purified proteins involved in transcription was developed for the investigation of specific interactions of proteins with radiolabeled DNA, RNA, ligands, and other small chemicals (22). A membrane-based high density array was developed for the purpose of screening a human fetal brain cDNA expression library consisting of 37830 clones. Purified proteins were spotted onto PVDF membranes at a density of 300 samples/cm2 (23). Other filter based arrays were constructed but the limitations were the low resolution and considerable background making it difficult to use them in applications with limiting sample quantities such as protein expression profiling of tumor biopsies.

Protein arrays are compromised of a library of proteins or antibodies immobilized in a 2D addressable grid on a chip (see Figure 1). Protein microarray biochips extract and retain targets from liquid media and are distinct from microfluidic biochips, which separate and process proteins in a transport medium in situ using microfluidic devices (24,25). A typical array may contain 103-104 spatially distinct elements within a total area of 1 cm2 (26).

Next: Types of Antibody and Protein Chips

References for Protein and Antibody Microarrays


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