Frozen protein arrays: a new method for arraying and detecting recombinant and native tissue proteins.
DNA microarrays are powerful tools for high throughput analysis of gene expression; however, they do not measure protein expression. Current methods for producing protein arrays require sophisticated equipment or extensive protein modification. We developed a low overhead, customizable assay platform called frozen protein arrays that can detect native proteins in protein lysates. Frozen protein arrays were formed from a block of frozen histologic embedding compound containing an array of wells. The wells were filled with samples, which freeze and bond to the block. Cryosections were cut and transferred to nitrocellulose-coated slides. The reproducibility, linearity, and sensitivity was confirmed using frozen protein arrays filled with prostate specific antigen. Frozen protein arrays could detect native tissue proteins. The alpha1 subunit of NaK-ATPase was detected in rat kidneys with a coefficient of variation of 4.3-6.6%. Frozen protein array analysis indicated that the protein abundance decreased by 48.7% following renal ischemia, similar to the 40% decrease by Western blotting. We conclude that frozen protein arrays are a low cost, moderate size platform for arraying samples including protein lysates. Production of many identical frozen protein arrays is easy, inexpensive, and requires only small sample volumes. The method is gentle on proteins as they remain frozen during production.
Miyaji
Takehiko
T
Renal Diagnostics and Therapeutics Unit, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-1268, USA.
Hewitt
Stephen M
SM
Liotta
Lance A
LA
Star
Robert A
RA
D000818:Animals; D005615:Freezing; D006801:Humans; D007511:Ischemia / Q000378:metabolism; D007668:Kidney / Q000201:enzymology; D017430:Prostate-Specific Antigen / Q000032:analysis / Q000276:immunology; D040081:Protein Array Analysis / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000032:analysis* / Q000276:immunology; D015203:Reproducibility of Results; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis; D014020:Tissue Extracts / Q000737:chemistry*
D011994:Recombinant Proteins; D014020:Tissue Extracts; D017430:Prostate-Specific Antigen; D000254:Sodium-Potassium-Exchanging ATPase