SDS-PAGE separates proteins based on their molecular size. Proteins are treated with the detergent SDS, which denatures them and coats them with a uniform negative charge. This ensures that movement through the gel matrix is determined almost entirely by mass. Standard SDS-PAGE, known as the Laemmli system, is effective for separating medium to large proteins, typically 20 to 250 kilodaltons (kDa). However, for very small proteins or peptides, generally under 20 kDa, the standard method fails to provide adequate resolution.
Tricine SDS-PAGE is a specialized modification engineered to overcome this limitation and deliver high-resolution separation for low molecular weight species, often down to 1 kDa. By altering the chemical composition of the buffer system, this method ensures that small proteins and peptides are sharply focused and separated without diffusing. This modified system is the preferred choice for researchers studying peptides, hormones, and smaller protein subunits.
Why Standard SDS-PAGE Fails for Small Proteins
The conventional Laemmli system uses a discontinuous buffer arrangement with a stacking gel and a separating gel to concentrate proteins. This system uses chloride ions as the fast-moving leading ion and the amino acid glycine as the slower trailing ion. The difference in mobility between these ions creates a voltage gradient that forces all proteins into a tight band, or stack, before they enter the separating gel.
In the stacking gel (pH 6.8), glycine is mostly neutral and migrates slowly behind the negatively charged proteins. When the proteins and ions reach the higher pH of the separating gel (pH 8.8), glycine becomes significantly more negatively charged. This higher charge causes the glycine to speed up, overtake the proteins, and break the stacking effect, allowing separation by size.
For very small proteins, a problem arises because free SDS detergent molecules, which are much smaller than the proteins, accumulate at the stacking front. In the standard system, these small proteins often run along with the leading edge of the SDS micelles. This results in fuzzy, poorly resolved bands or complete diffusion, making separation ineffective for proteins under 10–20 kDa.
The Chemical Mechanism of Tricine SDS-PAGE
The Tricine SDS-PAGE system solves the poor resolution problem by changing the trailing ion from glycine to Tricine. Tricine has a lower pKa value (8.15) compared to glycine (9.6). This difference allows Tricine to maintain a higher negative charge and higher mobility at the pH used in the separating gel.
The chloride ion remains the leading ion, but Tricine acts as a more efficient trailing ion for low molecular weight species. When ions and proteins move into the separating gel, the Tricine ions migrate at a speed that separates them effectively from the small proteins and peptides. This faster migration ensures that small proteins are separated from the zone of stacked SDS micelles and the buffer front.
This modification produces exceptionally sharp bands for proteins in the 2–20 kDa range. Proteins larger than about 30 kDa are destacked sooner in this system, ensuring a smooth passage into the separating gel and reducing potential overloading effects.
Operational Setup and Buffer Systems
Implementing Tricine SDS-PAGE requires specific modifications to the gel and buffer formulations compared to the standard Laemmli method. The gel matrix is prepared with a higher concentration of acrylamide to provide the finer sieving effect necessary for separating very small molecules. For separating peptides under 10 kDa, gels with acrylamide concentrations of 16.5% or higher are commonly used, sometimes including urea (4–8 M) to enhance separation and sharpen bands.
The gel components and buffers are precisely formulated to maintain the discontinuous pH and ion mobility necessary for separation. The separating gel buffer typically uses 3.0 M Tris at a pH of 8.45, while the stacking gel uses a lower concentration of Tris at a pH around 6.8. This specific pH difference, combined with Tricine, optimizes ion mobility for small protein separation.
Running Buffers
The running buffers utilize separate formulations for the cathode (upper tank) and anode (lower tank), which is a significant change from standard SDS-PAGE.
The cathode buffer, placed in the upper reservoir, contains Tris, SDS, and the critical component Tricine, typically at 0.1 M Tricine and a pH around 8.25.
Conversely, the anode buffer in the lower reservoir contains only Tris at a slightly higher pH, such as 8.9. This precise buffer composition and pH gradient are necessary for the Tricine system to function correctly and achieve high resolution.
Detection Methods for Low Molecular Weight Species
Visualizing the separated low molecular weight proteins and peptides presents a challenge due to their size and tendency to diffuse or be lost during processing. Traditional Coomassie Brilliant Blue staining often provides insufficient sensitivity for small proteins because they bind less dye than larger proteins. Furthermore, their small size means peptides can easily wash out of the gel during fixing and destaining steps.
More sensitive detection methods are employed to ensure visualization of the separated bands:
- Silver staining: Offers significantly higher sensitivity by reacting directly with the protein.
- Negative staining: Methods like zinc-based staining allow for rapid visualization and can be reversed for subsequent analysis.
For targeted detection, Western blotting requires specific optimization for peptides. Small proteins can easily pass straight through the membrane during the transfer step. This requires using membranes with smaller pore sizes, such as 0.22 \(\mu\)m polyvinylidene fluoride (PVDF). Additionally, the transfer time must be significantly shortened, often to only 15–20 minutes in a semi-dry system, to prevent the small peptides from being driven completely through the detection membrane.