Two-Dimensional Polyacrylamide Gel Electrophoresis: A Separation Technique for Analysis of Proteins in Complex Protein Mixture Based on Charge and Molecular Weight

Abstract

Source: Zhan, X., et al. Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome J. Vis. Exp. (2018)

In this video, we demonstrate a two-dimensional gel electrophoresis method that separates individual proteins from a complex protein mixture obtained from tumor tissues of human nonfunctional pituitary adenoma (NFPA). The proteins are separated based on their electric charge in the first dimension and further separated based on molecular weights in the second dimension.

Protocol

1. Preparation of Samples

1. Collect pituitary adenoma tissues (0.2 - 0.5 mg) from the neurosurgical department. Immediately freeze in liquid nitrogen, and then transfer to -80 °C for storage.
2. Add 2 mL of 0.9% NaCl in deionized distilled water (ddH₂O). Use this solution to lightly wash the blood from the tissue surface (3x).
   NOTE: ddH₂O with a conductivity of 18.2 MΩ/cm is used throughout the protocol. Some of the tissue might be lost when washing off blood from a tissue's surface.
3. Add a volume (10 mL; 4 °C) of the solution containing 2 M acetic ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ettan IPGphor 3	GE Healthcare		Isoelectric focusing system.
Ettan DALT II System	Amersham Pharmacia Biotech, Piscataway, NJ, USA		The vertical electrophoresis system
Ettan IPGphor strip holder	Amersham Pharmacia Biotech, Piscataway, NJ, USA
Ettan DALTsix multigel caster	Amersham Pharmacia Biotech, Piscataway, NJ, USA		Multigel caster
KimWipe	Kimvipe		Insoluble paper towel
Water	Made by PURELAB flex instrument
Polytron Model P710/35 homogenizer	Brinkmann Instruments, Westbury, NY
Pierce BCA Protein Assay Kit	Thermo Fisher Scientific	23227
2-D Quant Kit	GE Healthcare	80-6483-56
BIS-ACRYLAMIDE	AMRESCO	0172
ACRYLAMIDE	AMRESCO	0341
DTT	Sigma-Aldrich	D0632
Thiourea	Sigma-Aldrich	T8656
Urea	VETEC	V900119
SDS	AMRESCO	0227
CHAPS	AMRESCO	0465
TEMED	AMRESCO	M146
Ammonium Persulfate	AMRESCO	M133
IPG buffer pH 3-10, NL	GE Healthcare	17-6000-87
Immobiline Dry Strip pH 3-10NL, 18cm	GE Healthcare	17-1235-01