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

Published: April 30, 2023

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 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. 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…

Disclosures

The authors have nothing to disclose.

Materials

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

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Cite This Article
Two-Dimensional Polyacrylamide Gel Electrophoresis: A Separation Technique for Analysis of Proteins in Complex Protein Mixture Based on Charge and Molecular Weight. J. Vis. Exp. (Pending Publication), e21119, doi: (2023).

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