![]() ![]() With the increasing need for high throughput homogenizers in applications such as in screening strategies, various approaches were developed to increase the throughput of homogenization. Because sonication generates a significant amount of heat, it is important that this procedure should be carried out on ice using multiple short bursts. Although very effective at disrupting cells and partly homogenized tissue, sonication is not very effective for solid tissue. These sound waves can disrupt most cell samples in seconds. Sonication utilizes high frequency sound waves created by a probe that rapidly enlarges and contracts at high frequencies. ![]() In some cases, to achieve higher yield of target proteins that are expressed at lower levels, additional sonication of samples is done to disrupt cellular membranes. Glass-teflon homogenizers, Dounce hand homogenizers, or polytron homogenizers are generally used to mince tissues to release target proteins in homogenization buffer. ![]() The shearing force of Dounce homogenizers depends on the diameters of the pestle and container. Dounce homogenizers are inexpensive and effective at mildly lysing cells. If subcellular fractions are needed, simple cutting of tissue with scissors, then use of a handheld homogenizer for course grinding and subsequent use of glass Dounce homogenizer for shearing could be utilized. If active proteins are needed for subsequent native gel electrophoresis the homogenization procedure should avoid processes that generate heat or cause foaming. The method of sample preparation depends on desired use of the sample. Īlthough an ideal disruption method would involve only one step, many laboratories utilize two or more methods to obtain efficient sample disruption. Homogenization techniques (mechanical, sonication, chemical) employed for extraction of target proteins vary with types of tissues (liver, kidney, muscle, heart) or cells (suspended or cultured primary cells or cell lines). Moreover, multiple freeze/thaw cycles of protein samples must be avoided to prevent protein degradation. To improve western blot accuracy, samples should be quickly harvested in ice cold buffer (neutral pH) and should be immediately frozen in liquid nitrogen and stored at −80 ☌ until use to avoid proteolytic degradation by endogenous proteases (as sometimes indicated by gel streaks). Tissues are more complex than cultured cells and require more mechanical intervention than cultured cells for effective sample preparation. Some lysis buffers resulted in incomplete protein extraction of certain protein groups such as membrane proteins, proteasome subunits, and ribosomal proteins. A report by Glatter et al., 2015 showed that the detected total proteome mass of a sample was highly dependent on the sample preparation protocol. The principal factors that can influence sample processing include the time between tissue collection and its freezing, frequent freeze/thaw cycles of tissue samples, poor homogenization techniques, and use of inappropriate lysis buffer. The expression, conformation, and stability of proteins varies significantly with the buffer and experimental conditions utilized. To isolate an intracellular protein the cell membranes must be ruptured to free the cellular contents using a suitable lysis buffer to obtain a high yield of solubilized target protein. 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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