It is recommended to add TEMED and APS solution as final ingredients, as they initiate gel formation instantly. The total amount of protein loaded onto the gel must be optimized. underwent significant improvements with respect to the sensitivity, robustness and flexibility (4). At present, the western blot technique is usually widely used because of its specific advantages in comparison with other protein detection methods, such as mass spectrometry and enzyme-linked immunosorbent assay (ELISA). Overall, the immunoblot process has a great specificity and is cheap in comparison with mass spectrometry. Moreover, western blotting can detect proteins in small amounts of sample. On the other hand, semi-quantitative densitometric analysis is typically used to quantify protein expression on a blot, being less accurate than the ELISA detection method (5). Connexin (Cx) half-life is very short (i.e.1.5 to 5 h) compared to other transmembrane proteins (610). Connexin mRNA translation can be altered through microRNAs or RNA-binding proteins and may be altered through trafficking and degradation mechanisms (11,12). As such, 6 connexin proteins gather to form a hemichannel in a single cell and 2 hemichannels of adjacent cells can connect to establish space junctions (13). The latter are important communication channels between neighbouring cells, providing the passage of a number of molecules, such as cyclic adenosine monophosphate, adenosine triphosphate, inositol triphosphate, and ions, all which play important functions in liver homeostasis (1416). The liver was the first organ in which connexins have been identified and hence several research efforts over the last decades have been focused on the analysis of these particular space junction proteins (1720). In this regard, detection of connexin proteins using immunoblot analysis is a valuable method in liver research. In this chapter, an immunoblot process optimized for NVP-TAE 226 analysis of hepatic NVP-TAE 226 connexins, specifically Cx26, Cx32 and Cx43, is outlined. The procedure basically consists of 2 actions, namely sodiumdodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and NVP-TAE 226 immunoblotting. Adding SDS, a strong anionic detergent, to protein extracts will result in an equal distribution in chargeperunit mass. When applying voltage to the polyacrylamide gel, proteins will migrate at different velocity and will be separated according to their molecular excess Fam162a weight (21). Subsequently, separated proteins can be transferred onto a membrane, typically made of nitrocellulose, polyvinylidene difluoride or polyvinylpyrrolidone, upon establishment of an electrical field (22). In order to reduce nonspecific binding of antibodies around the membrane in the next step, blots are incubated in a solution made up of bovine serum albumin (BSA), nonfat dry milk or casein (23). Visualisation of the protein can be achieved by a number of methods, of which enhanced chemiluminescence (ECL) is the most popular one. ECL is usually a very sensitive NVP-TAE 226 method and can be used NVP-TAE 226 for relative quantification of protein abundance around the membrane by detecting horseradish peroxidase from antibodies. The reaction product produces luminescence (Fig. 1), which can be captured on photographic film, and this is usually correlated with the amount of target protein around the membrane (24). == Physique 1. == == 2. Materials == All solutions should be prepared with ultrapure water (i.e.purified and deionized water with a constant sensitivity of 18 M at 25C). == 2.1. Protein extraction == Ethylenediaminetetraacetic acid (EDTA) answer: 0.5 M EDTA in water. Adjust to pH 8.0 (seeNote 1). This answer can be stored for 6 months at 4C. Lysisbuffer (seeNote 2):Priorto use, add 100 L EDTA answer and 100 L 100x protease and phosphatase inhibitor cocktail to 9.8 mL radio-immunoprecipitation assay buffer. Centrifuge suitable for cooling at 4C. Sonicator (i.e.for cell culture samples) or mixer (i.e.for liver tissue samples). Rotator. == 2.2. Protein quantification assay (seeNote 3) == Bicinchoninic acid (BCA) working reagent:Priorto use, mix BCA reagent A with BCA reagent B in a ratio 50:1. The solution turns blue. BCA working reagent can be stored for several days in a closed container at room temperature. BSA standards (Table 1). Keep on ice. Flat-bottom 96-well plate. Multiplate reader. Incubator. == Table 1. == == 2.3. Preparation of gels (Table 2).
