TY - JOUR
T1 - 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
AU - Hachiya, Naomi S.
AU - Ohkubo, Takuya
AU - Kozuka, Yoshimichi
AU - Yamazaki, Mineo
AU - Mori, Osamu
AU - Mizusawa, Hidehiro
AU - Sakasegawa, Yuji
AU - Kaneko, Kiyotoshi
N1 - Funding Information:
We thank K. Watanabe and K. Takayama for technical assistance. This work was supported by grants from the Ministry of Health, Labor, and Welfare and the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and from the Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Agency.
PY - 2005/12/1
Y1 - 2005/12/1
N2 - 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.
AB - 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.
KW - Inclusion bodies
KW - Laser-microdissection
KW - Oligomeric Aip2p/Dld2p
KW - Phosphorylated tau
KW - Pick bodies
KW - Protein conformation unfolding activity
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U2 - 10.1016/j.ab.2005.09.004
DO - 10.1016/j.ab.2005.09.004
M3 - Article
C2 - 16229812
AN - SCOPUS:27744578531
VL - 347
SP - 106
EP - 111
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 1
ER -