Optimal protein extraction methods from diverse sample types for protein profiling by using Two-Dimensional Electrophoresis (2DE)
- Author:
Tan, A.A.
;
Azman, S.N.
;
Abdul Rani, N.R.
;
Kua, B.C.
;
Sasidharan, S.
;
Kiew, L.V.
;
Othman, N.
;
Noordin, R.
;
Chen, Y.*
- Publication Type:Journal Article
- From:Tropical Biomedicine
2011;28(3):620-629
- CountryMalaysia
- Language:English
-
Abstract:
There is a great diversity of protein samples types and origins, therefore the
optimal procedure for each sample type must be determined empirically. In order to obtain a
reproducible and complete sample presentation which view as many proteins as possible on
the desired 2DE gel, it is critical to perform additional sample preparation steps to improve
the quality of the final results, yet without selectively losing the proteins. To address this, we
developed a general method that is suitable for diverse sample types based on phenolchloroform
extraction method (represented by TRI reagent). This method was found to yield
good results when used to analyze human breast cancer cell line (MCF-7), Vibrio cholerae,
Cryptocaryon irritans cyst and liver abscess fat tissue. These types represent cell line,
bacteria, parasite cyst and pus respectively. For each type of samples, several attempts were
made to methodically compare protein isolation methods using TRI-reagent Kit, EasyBlue Kit,
PRO-PREPTM Protein Extraction Solution and lysis buffer. The most useful protocol allows the
extraction and separation of a wide diversity of protein samples that is reproducible among
repeated experiments. Our results demonstrated that the modified TRI-reagent Kit had the
highest protein yield as well as the greatest number of total proteins spots count for all type
of samples. Distinctive differences in spot patterns were also observed in the 2DE gel of
different extraction methods used for each type of sample.
- Full text:W020150615419765902395.pdf
