Some examples of genetic methods include conceptual translation, Site-directed mutagenesis, using a fusion protein, and matching allele with disease states.
[9] The goal is to obtain the protein in a pure form that retains its biological activity for further study, including functional assays, structural analysis, or therapeutic applications.
The purification process typically involves several steps, including cell lysis, protein extraction, and a combination of chromatographic and electrophoretic techniques.
The process often begins with cell lysis, where the cellular membranes are disrupted to release proteins into a solution.
Following lysis, the mixture is usually clarified by centrifugation to remove cell debris and insoluble material, allowing soluble proteins to be collected for further purification.
By gradually changing the ionic strength or pH of the elution buffer, bound proteins can be released in a controlled manner, allowing for effective separation.
A target protein is captured on a column containing a ligand that specifically binds to it, such as an antibody, enzyme substrate, or metal ion.
It often requires a careful choice of extraction buffers that contain salts, detergents, or stabilizers to preserve protein structure and activity.
Detergents such as Triton X-100 or SDS can be used to solubilize proteins from membranes by disrupting lipid bilayers, allowing for effective extraction.
Proteins are loaded onto a gel matrix, typically made of polyacrylamide or agarose, and an electric current is applied.
2D gel electrophoresis combines isoelectric focusing (IEF) and SDS-PAGE to achieve a high-resolution separation of proteins.
As an electric field is applied, proteins migrate until they reach the point where their net charge is zero, effectively focusing them into narrow bands.
Folin reagent is stable at only acidic conditions and the method is susceptible to skewing results depending on how much tryptophan and tyrosine is present in the examined protein.
[12] The Folin reagent binds to tryptophan and tyrosine which means the concentration of the two amino acids affects the sensitivity of the method.
Most proteins absorb light very well at 280 nanometers due to the presence of tryptophan and tyrosine, but the method is susceptible to varying amounts of the amino acids it relies on.