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.
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.
Hachiya
Naomi S
NS
Second Department of Physiology, Tokyo Medical University, Shinjuku-ku, Japan.
Ohkubo
Takuya
T
Kozuka
Yoshimichi
Y
Yamazaki
Mineo
M
Mori
Osamu
O
Mizusawa
Hidehiro
H
Sakasegawa
Yuji
Y
Kaneko
Kiyotoshi
K
D000368:Aged; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D015151:Immunoblotting / Q000379:methods*; D002479:Inclusion Bodies / Q000737:chemistry* / Q000276:immunology; D007770:L-Lactate Dehydrogenase / Q000737:chemistry*; D042844:L-Lactate Dehydrogenase (Cytochrome); D007834:Lasers; D008297:Male; D042282:Microdissection; D020774:Pick Disease of the Brain / Q000378:metabolism / Q000473:pathology; D011489:Protein Denaturation; D017510:Protein Folding; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D012680:Sensitivity and Specificity
D029701:Saccharomyces cerevisiae Proteins; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); C487563:DLD2 protein, S cerevisiae