![]() Aliquots of lysates equivalent to 80 μg of protein are electrophoresed through polyacrylamide gels and transferred to Hybond-P PVDF membranes (Amersham Biosciences, Buckinghamshire, UK). Protein concentrations are measured by using the DC protein assay method (BioRad, Hercules, CA, USA). The homogenates are sonicated and then centrifuged at 110 × g, and the resulting pellets are discarded. Katsuhiko Yoshizawa, in Animal Models for the Study of Human Disease (Second Edition), 2017 9.7 Western Blot Analysisįor western blot analysis, a protein extract is prepared by homogenizing the retinas in lysis buffer solution containing the protease inhibitors leupeptin, pepstatin, and aprotinin, each at a final concentration of 1 μg/mL. Northern blot analysis of rat tissues detected PGDS expression at high levels only in spleen and oviduct.Īiro Tsubura. The hematopoietic enzyme is the first recognized vertebrate homolog of the sigma class of glutathione S-transferase. Hematopoietic PGD synthase is widely distributed in the peripheral tissues and localized in the antigen-presenting cells, mast cells, and megakaryocytes. This enzyme is considered to be a dual-function protein it acts as a PGD2-producing enzyme and also as a lipophilic ligand-binding protein, because the enzyme binds retinoids, thyroids, and bile pigments, with high affinities. The human enzyme was identified as beta-trace, which is a major protein in human cerebrospinal fluid. It is secreted into cerebrospinal fluid, seminal plasma, and plasma, respectively. PTGDS is localized in the central nervous system and male genital organs of various mammals and the human heart. PDGS was not detected in human spleen and bone marrow. In humans, expression was highest in adipose tissue, macrophages, and placenta, with moderate levels in lung, a tissue in which expression was not detected in rat. Western blot analysis showed highest expression of PDGS in rat spleen and bone marrow. The obtained immunoprecipitated blot showed the reaction between antibody and P-gp by a dark band at 170 kDa, indicating the existence of the efflux protein P-gp in rabbit corneal epithelial layers and Statens Seruminstitut rabbit cornea cells.Īndreas Pahl, in xPharm: The Comprehensive Pharmacology Reference, 2007 Localization Protein Tween 80) and preferably a cocktail of protease inhibitors.Įxpression of P-gp in the Statens Seruminstitut rabbit cornea cell line (Statens Seruminstitut, Copenhagen, Denmark) and primary cultured rabbit corneal epithelial cells was determined with anti-P-gp monoclonal antibody by Dey et al. As the transporter proteins under investigation are membrane bound, the protein extraction buffer must contain surfactant (e.g. Preparation of the tissue sample is the key to the success of Western blotting. The use of specific protease inhibitors may also be considered. Ideally, ocular samples, as in the case of RT-PCR sample preparation, should be snap-frozen in liquid nitrogen immediately and preserved at approximately – 80 ☌ until further use. Careful precautions should be taken so that the proteins are not lost or changed as a result of proteolytic degradation. Biological states and sample quality should be preserved before samples are processed for Western blot/immunohistochemical analysis. However, similar to RT-PCR, care should be taken when preparing samples for Western blot analysis and immunohistochemical detection (described next). Unlike the PCR assay, Western blot analysis provides direct evidence for the presence of specific proteins. Subsequent to this step, staining is performed using specific antibodies. Then proteins are transferred to a sheet of specific blotting membrane (nitrocellulose or polyvinylidene fluoride), where the same pattern of separation will be retained. Proteins are separated by gel electrophoresis, usually sodium dodecyl sulfate–polyacrylamide gel electrophoresis, according to their size or three-dimensional structure. Western blot analysis is a widely used analytical technique to detect and measure specific proteins in different samples. Mitra, in Ocular Transporters and Receptors, 2013 3.2.2 Western blot analysis ![]()
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