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 |
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).