SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) is one of the most commonly used techniques in molecular biology and biochemistry. It allows proteins to be separated solely on the basis of their molecular weight.
How does SDS-PAGE work?
The principle behind electrophoresis is the movement of charged molecules in an electric field. The rate of migration during this process depends on three factors: the molecule’s charge, its size and its shape. Under native conditions, proteins differ in these respects.
The use of SDS denatures proteins (unfolding them) and gives them a uniform negative charge. This allows them to be separated by electrophoresis based solely on their molecular weight.
Properties of SDS (Sodium dodecyl sulphate)
SDS is an anionic detergent that is a key component of this method. It serves three functions: The structure of SDS
- Breaks non-covalent bonds: SDS breaks hydrogen bonds and hydrophobic interactions, causing protein denaturation.
- Masking the charge: SDS binds to proteins in a fixed mass ratio – approximately 1.4 g of SDS per 1 g of protein. The binding occurs with the hydrophobic regions of the protein. The negatively charged end of the SDS is directed outwards. The negative charge masks the natural charges of the amino acids. As a result, all proteins in the sample become negatively charged, and the charge-to-mass ratio remains constant for them.
- Linearization: Due to the repulsion of negative SDS groups, the polypeptide chain assumes a rod- or fibre-like structure.
Interaction between SDS and protein
Important: Proteins containing disulphide bonds will not denature completely unless they are reduced. To fully denature proteins containing disulphide bonds (S-S), a reducing agent such as β-mercaptoethanol or DTT (dithiothreitol) is added to the loading buffer.
3. How to prepare a sample for SDS-PAGE?
To prepare a sample for SDS-PAGE, you must first determine the protein content of the sample. To do this, you should use one of the techniques for determining protein content.
The sample must be diluted to the correct concentration. It must not be too dilute (protein bands at lower concentrations will be too faint or invisible) nor too concentrated (the bands will merge and bleed). Here are a few guidelines to follow:
The gel should be applied:
- 10–50 μg of protein from cell lysate
- 10–100 ng of purified protein
The typical volume dispensed into the wells is 5–35 μl.
The appropriately diluted sample is mixed with a loading buffer containing SDS and DTT or β-mercaptoethanol. The sample should be heated at 95 °C for a few minutes to reduce disulphide bonds. The loading buffer usually contains a dye, which allows the migration of the leading front of the electrophoretic separation to be tracked. Due to its glycerol or sucrose content, it has a high density, causing the sample to settle at the bottom of the well. Hence its common name – loading buffer.
Once cooled, the sample is placed in wells in a thickening gel.
4. Choosing the gel consistency
Polyacrylamide gel is formed by the polymerisation of acrylamide monomers cross-linked with bis-acrylamide. The pore size of the ‘sieve’ depends on the concentration of acrylamide.
The rule is simple:
- High acrylamide content (thick gel): Fine pores, ideal for the separation of small proteins.
- Low acrylamide concentration (low-viscosity gel): Large pores, ideal for the separation of large proteins.
| Acrylamid concentration(%) | Range of separation (kDa) | Exemplar application |
| 7-8% | 50 – 500 kDa | Large enzymes, protein complexes |
| 10% | 20 – 300 kDa | Universal range |
| 12-15% | 10 – 100 kDa | Typical cytosolic proteins |
| >15% | < 20 kDa | Small peptides |
It is worth bearing in mind the structure of a discontinuous gel (Laemmli system), which consists of two layers:
- Stacking gel: A top layer of thin gel with a lower pH (6.8) that compresses the sample into a narrow band. As the sample moves from the stacking gel to the separating gel, the proteins align in the correct order and become concentrated, resulting in thin, distinct bands.
- Resolving gel: The lower, denser gel with a higher pH (8.8), in which the actual separation by molecular weight takes place.
5. Construction of an electrophoresis apparatus
A standard SDS-PAGE kit consists of a vertical electrophoresis system.
The main elements are:
- Chamber (Tank): Filled with an electrolyte buffer, it provides cooling and electrical conductivity.
- Glass tiles: A gel is poured between them.
- Spacers: These determine the thickness of the gel (usually 0.75 mm – 1.5 mm).
- Comb: Creates wells into which samples are placed.
- Electrodes:
- Cathode (-): Located at the top (negative proteins are repelled by it).
- Anode (+): Located at the bottom (negative ions are attracted to it).
- Power supply: Generates an electric field (constant voltage or current).
6. Selection of SDS-PAGE separation conditions
The success of electrophoresis depends on the correct selection of current parameters and buffers.
- Electrode buffer: Tris-Glycine-SDS buffer is most commonly used. Glycine plays a key role in the process of protein stacking at the gel interface.
- Voltage (V):
- When passing through the densifying gel, a lower voltage (e.g. 80–100 V) is used to ensure that the proteins migrate evenly into the gel.
- In the insulating gel, the voltage increases (e.g. 120–150 V).
- Temperature: The flow of electric current generates heat (Joule’s law). If the temperature is too high, proteins migrate more rapidly towards the edges. As a result, so-called ‘smiling bands’ appear on the electropherogram. For this reason, electrophoresis apparatus is equipped with a cooling system (e.g. a flow-through system). If it does not have one, the separation can be carried out in a refrigerator.
7. Staining the gel
Once electrophoresis is complete, the proteins are invisible. They must be fixed and stained.
- Coomassie Blue (Coomassie Brilliant Blue R-250/G-250): The most common method. The dye binds non-specifically to proteins.
- Silver staining:
- Advantages: Very high sensitivity (detects as little as 1 ng of protein).
- Disadvantages: Time-consuming, requires the use of toxic reagents, difficult to reverse.
- Fluorescent staining (e.g. SYPRO Ruby): Requires a specialised scanner, but offers a wide linearity range (suitable for quantitative analysis).
8. Analysis of the results
To determine the size of the protein being analysed, a so-called size marker (protein ladder) – a mixture of proteins of known molecular weights – is applied to the same gel.
The analysis proceeds as follows:
- We compare the position of the band on our sample with the bands on the marker.
- For accurate calculations, the Rf value (protein migration distance / gel length) is determined.
- There is a linear relationship between the logarithm of the molecular weight (log Mw) and the migration distance.
Application
The SDS-PAGE technique is widely used in laboratories around the world. Its main applications include:
- Molecular weight estimation: Identification of unknown proteins based on their size.
- Assessing the purity of the sample: If only a single band is visible after separating the protein on a gel, the sample is pure. Multiple bands indicate contamination.
- Western blotting: SDS-PAGE is the first step in this technique (transferring proteins from the gel to a membrane for detection by antibodies).
- Monitoring protein expression: Comparison of cell lysates before and after protein production induction.
Literature
Berg, J. M., Tymoczko, J. L., Gatto, G. J., & Stryer, L. (2018). Biochemia. Wydawnictwo Naukowe PWN.
Hames, B. D. (1998). Gel electrophoresis of proteins: A practical approach. Oxford University Press.
Wilson, K., & Walker, J. (Eds.). (2010). Principles and techniques of biochemistry and molecular biology (7th ed.). Cambridge University Press.Przesuń wyżejPrzenieś w dółPrzełącz panel: Yoast SEOPrzesuń wyżejPrzenieś w dółPrzełącz panel: Języki