More than a 100-fold increase in immunoblot signals of laser-microdissected inclusion bodies with an excessive aggregation property by oligomeric actin interacting protein 2/D-lactate dehydrogenase protein 2.

Abstract

We established a histobiochemical approach targeting micron-order inclusion bodies possessing extensive aggregation properties in situ by using a nonchemical denaturant (oligomeric actin interacting protein 2/d-lactate dehydrogenase protein 2 [Aip2p/Dld2p]) with the combinatorial method of laser-microdissection and immunoblot analysis. As a model, pick bodies were chosen and laser-microdissected from three different brain regions of two patients with Pick's disease. Initially, 500 to 2000 pick bodies were applied onto SDS-PAGE gels after boiling in Laemmli's sample buffer according to established immunoblotting procedures; however, only faint signals were obtained. Following negative results with chemical denaturants or detergent, including 6 M guanidine hydrochloride, 8 M urea, and 2% SDS, the laser-microdissected pick bodies were pretreated with oligomeric Aip2p/Dld2p, which possesses robust protein unfolding activity under biological conditions. Strikingly, only one pick body was sufficient to illustrate an immunoblot signal, indicating that pretreatment with oligomeric Aip2p/Dld2p enhanced the immunoblot sensitivity by more than 100-fold. Pretreatment with oligomeric Aip2p/Dld2p also allowed us to quantify the total protein content of pick bodies. Thus, use of oligomeric Aip2p/Dld2p significantly contributed toward the acquisition of information pertaining to the molecular profile of proteins possessing an extensive aggregation property, particularly in small amounts.

• Authors: Naomi S Hachiya, Takuya Ohkubo, Yoshimichi Kozuka, Mineo Yamazaki, Osamu Mori, Hidehiro Mizusawa, Yuji Sakasegawa, Kiyotoshi Kaneko
• Journal: Analytical biochemistry (Anal Biochem) Vol. 347 Issue 1 Pg. 106-11 (Dec 01 2005) ISSN: 0003-2697 [Print] United States
• PMID: 16229812 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Saccharomyces cerevisiae Proteins
• L-Lactate Dehydrogenase
• L-Lactate Dehydrogenase (Cytochrome)
• DLD2 protein, S cerevisiae

Topics

• Aged
• Electrophoresis, Polyacrylamide Gel
• Female
• Humans
• Immunoblotting (methods)
• Inclusion Bodies (chemistry, immunology)
• L-Lactate Dehydrogenase (chemistry)
• L-Lactate Dehydrogenase (Cytochrome)
• Lasers
• Male
• Microdissection
• Pick Disease of the Brain (metabolism, pathology)
• Protein Denaturation
• Protein Folding
• Saccharomyces cerevisiae Proteins (chemistry)
• Sensitivity and Specificity