Western analysis

Figure 9. Western analysis of P-subunit and PG2 antigen levels during tomato flower...

Comparative Western analysis of samples directly extracted from tissue without intermediate purification [Pg.276]

Figure 4. Western analysis of culture filtrate of A. aculeatus wild type (lanes 1-3) and A. aculeatus (lanes 4-6) and A. awamori multicopy transformants (lanes 7-9). Medium samples were applied undiluted (lanes 1,4,7), 10 times diluted (lanes 2,5,8) or 100 times diluted (lanes 3,6,9). Lane 10 contains purified rhamnogalacturonase.

In addition, Western analysis can be used to verify that the tag has been efficiently added to the protein and that the resultant tagged protein s abundance is not altered by the presence of the tag. Using either an antibody specific to your protein of interested or an anti-GFP antibody (Clontech), a correctly tagged protein should be approximately 29 kDa heavier than the untagged protein (Fig. 3.ID). 

In the Drosophila circadian clock, three proteins are rhythmically phosphorylated throughout the circadian cycle PER, TIM and dCLK. The electrophoretic mobility of these three proteins all undergo changes during the circadian day by Western analysis. In all cases, phosphatase treatment reduced or eliminated the slower migrating bands, suggesting that the mobility shifts are due to phosphorylation (Edery et al 1994, Zeng et al 1996, Lee et al 1998). While there is evidence that DBT phosphorylates PER and SGG phosphorylates TIM, it is possible that other kinases phosphorylate these proteins as well. TIM, for instance, is phosphorylated by a tyrosine kinase before it is ubiquitinated and... 

Cycling protein phosphorylation plays a role in the mammalian circadian clock as well. PERI, PER2 and BMAL all show temporal changes in electrophoretic mobility that are eliminated by phosphatase treatment (Lee et al 2001). Although these PER phosphorylations are likely to reflect CKl activity they may not be the only clock-related substrates of this enzyme family. CRYl and CRY2, for instance, can be phosphorylated by CKls in vitro when present in a CRY/PER/CKle complex (Eide et al 2002). Two isoforms of mammalian CLOCK (orthologue of Drosophila CLK) also appear to be phosphorylated, resulting in mobility shifts by Western analysis (Lee et al 2001). The kinase(s) responsible for CLOCK phosphorylation is (are) unknown. 

Fessard, V. and Livingstone, D.R. (1998). Development of western analysis of oxidized proteins as a biomarker of oxidative damage in liver of fish. Marine Environmental Research, 46 407-410. 

Gotzmann, J. and Gemer, C. (2000) A method to produce ponceau replicas from blots application for western analysis. Electrophoresis 21, 523-525. 

Fig. 13.1 Phosphorylated c-Ski expression in cytosolic fraction of cells in post-MI rat heart. In post-MI rat hearts with chronic large myocardial infarction, phosphorylated c-Ski expression was noted in the cytosol of cells populating the infarct scar (predominantly myofibroblasts) and the remnant heart (mixed cytosolic fraction from myocytes and nonmyocytes). Hearts were sampled at different times after surgical ligation of coronary artery occlusion and Western analysis was carried out to assess phosphorylated c-Ski expression. Trends in -fold protein expression (target band intensity corrected for loading) are shown in curves.

Immunoprecipitation can also be carried out in gds, resulting in the formation of precipitated bands which can be visualised dther directly, or by staining. These approaches have been largely superseded by techniques based on ELISA and Western analysis. 

The polyclonal antiserum, obtained by immunisation of rabbits as described in Materials and Methods was initially used for immunoprecipitation of metabolically labelled primary cortical neurons, providing a major band at 110 kDa corresponding to intact neuronal APP (Figure 2 A). The antibody was also tested by western analysis yielding... 

The effects of balanol and 10 -deoxybalanol on intact cells have been exmined to determine whether these compounds cross the cell membrane or if the potency and specificity observed in vitro are present in vivo. Western analysis showed that both compounds reduced phosphorylation of CREB in isoproterenol-stimulated A431 cells (ICso = 3 jM), although only balanol inhibits phosphorylation of MARCKS protein in phorbol ester-stimulated A431 cells (IC50 = 7 pM) 109],... 

Figure 2 Characteristics of AR polymorphisms in transfected cells. (A) Agonist-promoted down-regulation phenotypes of the Ser49 and Gly49 ARs. (B) Western analysis of the Ser49 and Gly49 /SjARs. (C) Adenylyl cyclase activities of the Arg389 and Gly389 /SiARs. ISO = isoproterenol, Cyclo = cycloheximide.

The coimmunoprecipitation of proteins by an antibody is the standard procedure to demonstrate the presence of interacting proteins. In this method, when a protein is precipitated by a specific antibody, the interacting protein(s) are also precipitated along with the protein precipitated by the antigen-antibody interaction the components of interacting protein are then visualized by Western analysis within the same band on the gel after electrophoresis. 

B and C). A strong band was observed which is absent in the non-transgenic plant TP309 (Fig. 8.7 B) this band was confirmed to be ITF fusion by Western analysis using anti-ITF antibody (Fig. 

Glycosynthase activity for tetrasaccharide synthesis from a-lactosyl fluoride and p-nitrophenyl /3-cellobioside (PNPC) was measured for the Humicola insolens Cel7B variants in sodium phosphate buffer, pH 7.0, at room temperature. Specific activities were determined by measuring the fluoride ion release rate by a fluoride ion selective electrode. The protein purification yields are the yield of purified protein as determined by western analysis from total cell culture. 

Wash 3x 5 min in binding buffer at room temperature, expose to X-ray film overnight at -80°C. The probe can be stripped by incubating 3x 20 min in 100 ml of stripping buffer at room temperature. The same filter can then be washed in TST and used for Western analysis (blocking step is not necessary). This way the biotinylated marker can be visualized for size determination. 

Wash the beads four times with PBS containing 1% NP-40 (instead of PBS, RIPA buffer may be used for more stringent washing). Wash the beads two times with a buffer containing 10 mM Tris-HQ, pH 8.0, 0.25 M LiQ, 0.5% NP-40, 0.5% Na deoxycholate, 1 mM EDTA. Wash the beads two times with TE. Resuspend the beads in 120 fil of TE. Remove 10 /il for Western analysis. Remove 10 fil and count [ Hjthymidine. This will give an idea of how efficient the immunoprecipitation was, and should show a large difference between preimmune and anti-SATBl serum. 

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