Comparison of three methods of protein extraction from Dermatophagoides pteronyssinus for two-dimensional electrophoresis.
OBJECTIVE
To explore an effective method of Dermatophagoides pteronyssinus protein extraction suitable for two-dimensional electrophoresis (2-DE) analysis.


METHODS
The extracts of Dermatophagoides pteronyssinus were prepared with Coca's solution, lysis buffer of 2-DE, and Trizol reagent, respectively. Bicinchoninic acid (BCA) assay was used to determine the total protein concentration of the samples. The efficiency of different protein extraction methods were evaluated with 2-DE analysis.


RESULTS
The concentrations of extracted protein by methods of Coca's solution, lysis buffer, and Trizol reagent were 0.63 g/L, 0.90 g/L, and 0.80 g/L, respectively. The 2-DE analysis results showed that some protein spots in low molecular weight (LMW) range could be detected with the Coca's solution method. With the lysis buffer of 2-DE method, more protein spots in LMW range could be detected, while the medium molecular weight (MMW) protein spots were absent. Several MMW protein spots (174-178 kD and 133 kD) and more LMW protein spots were detected with Trizol reagent method.


CONCLUSIONS
Among Coca's solution, lysis buffer of 2-DE, and Trizol reagent, the concentration of extracted protein of Dermatophagoides pteronyssinus by lysis buffer of 2-DE is the highest. However, most protein components of Dermatophagoides pteronyssinus purified mite bodies can be extracted by Trizol reagent, which may generally reflect the whole profile of Dermatophagoides pteronyssinus allergens.
Sun
Jin-lu
JL
Department of Allergy, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China. Sunjl5@yahoo.com
Zhang
Hong-yu
HY
Guo
Zhi-yi
ZY
Ying
Wan-tao
WT
Qian
Xiao-hong
XH
Wang
Jing-lan
JL
D000485:Allergens / Q000302:isolation & purification; D000818:Animals; D040002:Dermatophagoides pteronyssinus / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D006146:Guanidines / Q000737:chemistry; D010636:Phenols / Q000737:chemistry; D011506:Proteins / Q000302:isolation & purification*
D000485:Allergens; D006146:Guanidines; D010636:Phenols; D011506:Proteins; C411644:trizol