Reagents SDS-PAGE
SDS-PAGE or Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis is a technique used for the separation of proteins based on their molecular weight. The different buffer and reagents with their purpose for vertical gel electrophoresis are as follows-
- N, N, N’, N’-tetramethylethylenediamine (TEMED) it catalyzes the acrylamide polymerization.
- Ammonium persulfate (APS) – it is an initiator for the acrylamide polymerization.
- Tris-HCl – it is the component of running and gel casting buffer.
- Glycine – it is the component of running buffer.
- Bromophenol blue – it is the tracking dye to monitor the progress of gel electrophoresis.
- Coomassie brilliant blue R250 – it is used to stain the polyacrylamide gel.
- Sodium dodecyl sulphate – anionic detergent used to denature and provide negative charge to the protein.
- Acrylamide – monomeric unit used to prepare the gel.
- Bis-acrylamide – cross linker for polymerization of acrylamide monomer to form gel.
Steps to denature proteins
Casting of the gel
- The acrylamide solution (a mixture of monomeric acrylamide and a bifunctional crosslinker bisacrylamide) is mixed with the TEMED and APS and poured in between the glass plate fitted into the gel caster.
- Ammonium persulfate in the presence of TEMED forms oxygen free radicals and induces the polymerization of acrylamide monomer to form a linear polymer.
- These linear monomers are interconnected by the cross linking with bis-acrylamide monomer to form a 3-D mesh with pores.
- The size of pore is controlled by the concentration of acrylamide and amount of bis-acrylamide in the gel.
- In a vertical gel electrophoresis system, we cast two types of gels, stacking gel and resolving gel.
- First the resolving gel solution is prepared and poured into the gel cassette for polymerization.
- A thin layer of organisc solvent (such as butanol or isoproponal) is layered to stop the entry of oxygen (oxygen neutralizes the free radical and slow down the polymerization) and make the top layer smooth.
- After polymerization of the resolving gel, a stacking gel is poured, and comb is fitted into the gel for construction of different lanes for the samples.
Running of the gel
- The sample is prepared in the loading dye containing SDS, β-mercaptoethanol in glycerol to denature the sample and presence of glycerol facilitates the loading of sample in the well.
- The pH of the stacking gel is 6.8 and at this pH, glycine is moving slowly in the front where as Tris-HCl is moving fast.
- As a result, the sample gets sandwiched between glycine-Tris and get stacked in the form of thin band. As the sample enters into the resolving gel with a pH 8.8, the glycine is now charged, it moves fast and now sample runs as per their molecular weight (due to SDS they have equal negative charge).
- After tracking dye reaches to the bottom of the gel, gel is taken out from the glass plate with the help of a spatula and it is stained with coomassie brilliant blue R250 dye.
- The dye stains protein present on the gel.
SDS PAGE Reagents Preparation
The acrylamide percentage in SDS PAGE gel depends on the size of the target protein in the sample.
Acrylamide % | M.W. Range |
7% | 50 kDa – 500 kDa |
10% | 20 kDa – 300 kDa |
12% | 10 kDa – 200 kDa |
15% | 3 kDa – 100 kDa |
For 5ml stacking gel:
H2O | 2.975 ml |
0.5 M Tris-HCl, pH 6.8 | 1.25 ml |
10% (w/v) SDS | 0.05 ml |
Acrylamide/Bis-acrylamide (30%/0.8% w/v) | 0.67 ml |
10% (w/v) ammonium persulfate (AP) | 0.05 ml |
TEMED | 0.005 ml |
For a 10ml separating gel:
Acylamide percentage | 6% | 8% | 10% | 12% | 15% |
H2O | 5.2ml | 4.6ml | 3.8ml | 3.2ml | 2.2ml |
Acrylamide/Bis-acrylamide (30%/0.8% w/v) | 2ml | 2.6ml | 3.4ml | 4ml | 5ml |
1.5M Tris(pH=8.8) | 2.6ml | 2.6ml | 2.6ml | 2.6ml | 2.6ml |
10% (w/v)SDS | 0.1ml | 0.1ml | 0.1ml | 0.1ml | 0.1ml |
10% (w/v) ammonium persulfate (AP) | 100μl | 100μl | 100μl | 100μl | 100μl |
TEMED | 10μl | 10μl | 10μl | 10μl | 10μl |
5X Sample buffer (loading buffer):
10% w/v | SDS |
10 mM | Dithiothreitol, or beta-mercapto-ethanol |
20 % v/v | Glycerol |
0.2 M | Tris-HCl, pH 6.8 |
0.05% w/v | Bromophenolblue |
1x Running Buffer:
25 mM | Tris-HCl |
200 mM | Glycine |
0.1% (w/v) | SDS |
References
- goldbio.com ↩︎
- Yang, Jae-Hyuk & Lim, Yoon-Kyu. (2011). Characteristics on Equine Herpesvirus Type 3 from Korea. Journal of Life Science. 21. 10.5352/JLS.2011.21.8.1156. ↩︎
- SDS-PAGE (assay-protocol.com) ↩︎