2.2.1 Denaturing Gel SDS-PAGE
Protein Denaturation and SDS-PAGE Related Protocols
Proteins naturally fold into various conformations, which affect their mobility in separation media such as gels.
Denatured proteins can counteract these structural effects, enabling separation that accurately reflects the protein's mass-to-charge ratio.
To denature proteins, the detergent sodium dodecyl sulfate (SDS) is typically added in gel electrophoresis.
SDS is simply added to the sample and is also a component of both the gel and the running buffer.
1.4 grams of SDS binds to each gram of protein, so any inherent charge on the protein is masked by the negatively charged detergent micelles.
Denaturing gels can be run under non-reducing conditions (without sample boiling or the addition of reducing agents), which is important for preserving the native structure of proteins for further analysis.
Alternatively, denaturing gels can be run under reducing conditions, where reducing agents such as dithiothreitol (DTT) or β-mercaptoethanol are added to the sample buffer and the mixture is heated.
These reagents disrupt the quaternary and tertiary structures of proteins by cleaving disulfide bonds between cysteine residues, resulting in linear polypeptide chains. Proteins treated in this way migrate at a rate that is a linear function of the logarithm of their molecular weight.
The differences between denaturing gel SDS-PAGE and the other two electrophoresis methods are as follows:
Denaturants such as SDS need to be added to the gel, and the sample also needs to undergo denaturation treatment (e.g., boiling). This denatures the protein and dissociates its subunits, making it impossible to reflect the true molecular weight of proteins containing subunits, nor to perform subsequent protein activity assays.
The separation of protein bands is mainly based on the molecular weight of the proteins.
Reagent Formulations and Membrane Transfer Reference Times
1. 1M Tris-HCl (pH 6.8), 1L:
Weigh 121.1g of Tris and place it in a 1L beaker. Add approximately 800ml of deionized water and stir thoroughly to dissolve.
Add concentrated hydrochloric acid (HCl) to adjust the pH to 6.8. (Reference: Approximately 70ml of concentrated HCl is required to reach pH 7.4.)
Dilute the solution to a final volume of 1L.
After autoclaving (high temperature and high pressure sterilization), store at room temperature.
Note: The solution should be cooled to room temperature before adjusting the pH, as the pH of Tris solutions varies significantly with temperature—for every 1°C increase in temperature, the pH of the solution decreases by approximately 0.03 units.
2. 1.5M Tris-HCl (pH 8.8), 1L:
Weigh 181.7g of Tris and place it in a 1L beaker. Add approximately 800ml of deionized water and stir thoroughly to dissolve.
Add concentrated HCl to adjust the pH to 8.8. (Reference: Approximately 42ml of concentrated HCl is required to reach pH 8.0.)
Dilute the solution to a final volume of 1L.
After autoclaving, store at room temperature.
Note: The solution should be cooled to room temperature before adjusting the pH, as the pH of Tris solutions varies significantly with temperature—for every 1°C increase in temperature, the pH of the solution decreases by approximately 0.03 units.
3. 10% (W/V) SDS, 100ml:
Weigh 10g of high-purity SDS and place it in a 100–200ml beaker. Add approximately 80ml of deionized water and heat at 68°C to dissolve.
Add concentrated HCl dropwise to adjust the pH to 7.2.
Dilute the solution to a final volume of 100ml, then store at room temperature.
4. 5×Tris-Glycine Buffer (SDS-PAGE Running Buffer), 1L:
Reagent Mass
Tris 15.1g
Glycine 94g
SDS 5.0g
Add approximately 800ml of deionized water and stir to dissolve.
Dilute the solution to a final volume of 1L and store at room temperature.
Note: The glass plates used for gel casting must be thoroughly cleaned. All buffers should be freshly prepared, and their pH values must be accurate.
5. Homemade Gel Concentration and Membrane Transfer Time (Reference Table):
Molecular Weight (KDa) Gel Concentration Transfer Time (250mA, h)
140—200 6% 2.0—3.0
80—140 8% 1.5—2.0
25—80 10% 1.5
15—40 12% 0.75
<20 15% 0.5
