Polyacrylamide Gel Electrophoresis: An Analytical Technique to Detect Heparan Sulfates of Varying Chain Lengths Isolated from Mouse Lung Tissue
Published: April 30, 2023
Abstract
Source: LaRiviere, W. B. et al., Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining. J. Vis. Exp. (2021)
This video demonstrates the separation procedure of heparan sulfates isolated from mouse lung tissue using polyacrylamide gel electrophoresis.
Protocol
1. Polyacrylamide gel electrophoresis of isolated and purified glycosaminoglycans Prepare solutions necessary for polyacrylamide gel electrophoresis (PAGE) in advance (Table 1). NOTE: Select the percent acrylamide of resolving gel solution depending on the size of the glycosaminoglycans expected to be in the sample. 15% is recommended for resolving larger fragments (greater than 30 disaccharide subunits in length); 22% for…
Representative Results
Table 1: Solutions required for polyacrylamide gel electrophoresis of purified glycosaminoglycans. All solutions must be filtered (0.22 µm) before use. NOTE: All solutions must be filtered (0.22μm) before use. Solution Recipe Composition …
Declarações
The authors have nothing to disclose.
Materials
| Ammonium persulfate (solid) | ThermoFisher Scientific | BP179-25 | Electrophoresis grade |
| Barnstead GenPure Pro Water Purification System | ThermoFisher Scientific | 10-451-217PKG | Any water deionizing/ purification system is an acceptable substitute |
| Boric acid (solid) | ThermoFisher Scientific | A73-500 | Molecular biology grade |
| Bromphenol blue (solid) | ThermoFisher Scientific | B392-5 | |
| Criterion empty cassette for PAGE (1.0mm thick, 12+2 wells) | Bio-Rad | 3459901 | Any 1.0mm thick PAGE casting cassette system will suffice |
| Criterion PAGE Cell system (cell and power supply) | Bio-Rad | 1656019 | Any comparable vertical gel PAGE system will work) |
| EDTA disodium salt (solid) | ThermoFisher Scientific | 02-002-786 | Molecular biology grade |
| Glycine (solid) | ThermoFisher Scientific | G48-500 | Electrophoresis grade |
| Hydrochloric acid (liquid) | ThermoFisher Scientific | A466-250 | |
| N,N'-methylene-bis-acrylamide (solid) | ThermoFisher Scientific | BP171-25 | Electrophoresis grade |
| Phenol red (solid) | ThermoFisher Scientific | P74-10 | Free acid |
| Sucrose (solid) | ThermoFisher Scientific | BP220-1 | Molecular biology grade |
| TEMED (N,N,N',N'-tetramethylenediamine) | ThermoFisher Scientific | BP150-20 | Electrophoresis grade |
| Tris base (solid) | ThermoFisher Scientific | BP152-500 | Molecular biology grade |
| Vacuum filter unit, single use, 0.22uM pore PES, 500mL volume | ThermoFisher Scientific | 569-0020 | Alternative volumes and filter materials acceptable |
| Acrylamide (solid) | ThermoFisher Scientific | BP170-100 | Electrophoresis grade |
| Heparin derived decasaccharide (dp10) | Galen scientific | HO10 | |
| Heparin derived hexasaccharde (dp6) | Galen scientific | HO06 | |
| Heparin derived oligosaccharide (dp20) | Galen scientific | HO20 |
Citar este artigo
Polyacrylamide Gel Electrophoresis: An Analytical Technique to Detect Heparan Sulfates of Varying Chain Lengths Isolated from Mouse Lung Tissue. J. Vis. Exp. (Pending Publication), e21106, doi: (2023).