Isolate Proteins That Bind To Antihis6 Antibody

1. RUN EACH SAMPLE ON SDS-PAGE

2. TRANSFER TO NITROCELLULOSE

3. CUT TWO LANES APART AND DO WESTERN BLOT WITH OPPOSITE ANTIBODY

Add antibody to HIS

Add antibody to HIS

Add antibody to FLAG

Add antibody to FLAG

Protein arrays can be constructed if all the proteins carry tags that allow binding to the array support.

Protein Arrays

Previous studies of proteins generally examined a single protein at a time. With the recent sequencing of whole genomes, proteome analysis has turned to methods that allow simultaneous monitoring of multiple proteins. Microarrays have been used for DNA for some time, but the variable structures and properties of proteins made such an array approach more difficult. Nonetheless, new technologies have been developed that allow high-throughput analysis of proteins. As a result, protein microarrays have recently become available for proteome analysis.

Protein microarrays have been used for the biochemical and enzymatic analysis of proteins as well as to survey protein-protein interactions. So far most proteome arrays have used the yeast, Saccharomyces cerevisiae, as model organism. A complete pro-teome analysis needs an array of approximately 6,000 proteins in this case. Such arrays are assembled using proteins that have been tagged with groups allowing binding to solid supports such as 96-well microtiter dishes or glass microscope slides.

Libraries that include nearly 90% of the yeast proteins have been fused to the glutathione-S-transferase (GST) tag, which allows binding to a solid support via glutathione or to the His tag, which allows binding via nickel (Fig. 26.23). These constructs have been expressed under control of the GAL1 (galactose inducible) or CUP1 (copper inducible) promoters. Such protein libraries may be pooled or distributed individually into the wells of microtiter dishes. Simpler and less expensive screening is usually done individually, whereas complex or expensive assays are more often run first on pooled protein samples that are subdivided for further analysis if positive results are found.

The functional assays must be designed so that the arrays can be screened conveniently, usually for fluorescence, less often for radioactivity. For example, the yeast proteome has been screened for those proteins that bind calmodulin (a small calcium binding protein) or phospholipids, in the laboratory of Michael Snyder at Yale University. The His-tagged proteins were attached to nickel-coated glass slides. Both calmodulin and phospholipid were tagged with biotin. After binding of calmodulin or phospholipids to the proteome array, the biotin was detected by streptavidin carrying a Cy3 fluorescent label (Fig. 26.24). This revealed 39 calmodulin-binding proteins of which six were previously known. Some 150 phospholipid-binding proteins were also found.

Global surveys of low molecular weight metabolites can be done with NMR or high resolution mass spectrometry.

Metabolomics

By analogy with genome and proteome, the metabolome is the totality of small molecules and metabolic intermediates. NMR of extracts from cells labeled with 13C-glucose has allowed simultaneous measurement of multiple metabolic intermediates. An alternative is the separation of metabolites from cells labeled with 14C-glucose by thin layer chromatography. However, these methods are limited in both sensitivity and in the number and chemical types of compounds that can be readily separated.

Nearly complete metabolome analysis may be achieved by mass spectrometry. This approach is not limited to particular classes of molecule and is extremely sensitive. Carbon-12 is defined as having a mass of exactly 12 daltons. However, the masses of other atoms, such as 14N or 16O, are not exact integers. Consequently mass spectrome-try using extremely high mass resolution (EHMR; that is to 1 ppm or less) allows the unambiguous determination of the molecular formula of any metabolite. Isomers have the same molecular formula, but may be distinguished by the different fragmentation patterns of their molecular ions.

calmodulin A small calcium binding protein of animal cells metabolome The total complement of small molecules and metabolic intermediates of a cell or organism protein microarray Microarray of immobilized proteins used for proteome analysis and normally screened by fluorescent or radioactive labeling

FIGURE 26.23 Protein Microarray—Principle

To assemble a protein microarray, a library of His-tagged proteins is incubated with a nickel coated glass slide. The proteins adhere to the slide wherever a nickel ion is attached.

FIGURE 26.24 Screening Protein Microarray using Biotin/Streptavidin

Protein microarrays can be screened to find proteins that bind to phospholipids, for example. The protein microarray is incubated with phospholipid bound to biotin. Then the bound phospholipid is visualized by adding avidin conjugated to a fluorescent dye. Spots that fluoresce represent specific proteins that bind phospholipids.

Phospholipid tagged with biotin

Avidin with cy3 fluorescent label

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