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

Naomi S. Hachiya, Takuya Ohkubo, Yoshimichi Kozuka, Mineo Yamazaki, Osamu Mori, Hidehiro Mizusawa, Yuji Sakasegawa, Kiyotoshi Kaneko

Research output: Contribution to journalArticlepeer-review

6 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)106-111
Number of pages6
JournalAnalytical Biochemistry
Volume347
Issue number1
DOIs
Publication statusPublished - 2005 Dec 1

Keywords

  • Inclusion bodies
  • Laser-microdissection
  • Oligomeric Aip2p/Dld2p
  • Phosphorylated tau
  • Pick bodies
  • Protein conformation unfolding activity

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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