Electrophoresis

The 20X SSC Buffer, with a pH between 6.9 and 7.1, is essential for in molecular biology, particularly for Southern and Northern hybridization techniques. This solution is sterilized through a 0.22 µm filter, making it suitable for direct use or for dilution based on experimental requirements. In parallel, 20X SSPE is available as a concentrated buffer intended for nucleic acid hybridizations and blot transfer processes. A significant feature of SSPE is its inclusion of EDTA, which chelates divalent metal ions like Mg²+. This property effectively inhibits DNase activity, helping to preserve the concentrations of probe and target DNA during Southern hybridization, thereby enhancing the accuracy of the results.

The Tris-Tricine-SDS Gel Running Buffer is specifically designed for separating proteins with molecular weights between 1 and 100 kDa, making it particularly effective for resolving proteins smaller than 30 kDa. This approach differs from traditional SDS-PAGE by substituting glycine (pK 9.6) with tricine (pK 8.15), which enhances the stacking and destacking of low molecular weight proteins and improves the resolution of smaller peptides due to the differing pK values. Incorporating urea into the stacking gel allows for the effective separation of two proteins that share the same molecular weight. Additionally, the lower acrylamide concentrations in Tricine gels facilitate the transfer of hydrophobic proteins during Western blotting. Tricine–SDS-PAGE is therefore commonly employed for the separation of lower molecular weight proteins.

SDS-PAGE is a method employed for separating proteins via electrophoresis, based on the principle that charged molecules migrate through a matrix in response to an applied electric field. The matrix utilized for this separation is polyacrylamide, which is derived from acrylamide, a compound that can be hazardous. Polyacrylamide is commonly used for the size-based separation of proteins and nucleic acids. The gel matrix forms through the free radical polymerization of acrylamide along with a crosslinking agent known as N, N-Methylenebisacrylamide. In this process, acrylamide monomers polymerize into long chains, initiated by a free radical-generating system, and these chains are linked by the cross-linker, resulting in a gel structure. This gel is crucial for effective electrophoretic separation, facilitating the analysis of biomolecules based on size.