Western blot hiv specificity12/16/2023 Protein samples used in western blot are very diverse, ranging from purified proteins to highly complex samples such as cell/tissue lysates that contains cellular debris, protein aggregates, fats and proteases. Thus, care must be taken to standardize each step of the western blot process for reproducibility and high sensitivity.ģ. Improvement in protein purification over last decade Variations at any step of the process can influence the result. It is a multistep procedure ( Figure 1) that typically involves a) sample preparation (protein extraction and measurement of protein concentration) from cells or tissue lysates, b) separation of proteins by size on sodium dodecyl sulphate (SDS) polyacrylamide gel by electrophoresis, c) immobilization of separated proteins in a nitrocellulose or polyvinylidene fluoride (PVDF) membrane, d) blocking of non-specific proteins on membrane, e) probing of target proteins with specific primary antibodies, e) incubation with secondary antibody conjugated with labelled chemiluminescent or fluorescent molecule, f) detection of signal that reflects antigen/ antibody binding, g) densitometry analysis of protein bands of interest using a software. Western blotting is an extensively used technique for protein analysis. The western blot is a common method used to detect proteins as well as post-translational modifications on proteins, and can provide semi-quantitative or quantitative data about the target protein in simple or complex biological samples.
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