Western blot fractions

A. Western blot analysis of membrane fractions from P. vulgaris probed with a PGIP-specific antibody. 1) PGIP purified from P. vulgaris] 2) and 3) membranes 4) supernatant of the membrane preparation. 

The PemB cellular localisation was determined both in E. chrysanthenu and in an E. coli recombinant strain by Western blot of the cell fractions with a PemB-antiserum. No PemB was detected in the culture supernatant and only trace amounts were found in the soluble cell fractions - periplasm and cytoplasm (Figure 2). PemB was found mostly in the total membrane fraction from which it could be completely extracted by Triton X-100/Mg2+ and partially extracted by Sarkosyl (Figure 2). This behaviour is typical of inner membrane proteins, but since some exceptions have been noticed it does not positively indicate the PemB localisation (15). We performed cell membrane fractionation in sucrose density gradient centrifugation both by sedimentation and flotation, using several markers of inner and outer membrane vesicles. PemB was found in the outer membrane vesicles (data not shown). 

Because the protein analyte is endogenous to the plant, it can be difficult to demonstrate the efficiency of the extraction procedure. Ideally, an alternative detection method (e.g., Western blotting) is used for comparison with the immunoassay results. Another approach to addressing extraction efficiency is to demonstrate the recovery of each type of protein analyte from each type of food fraction by exhaustive extraction, i.e., repeatedly extracting the sample until no more of the protein is detected. " ... 

The entire eluted fraction and the aliquots containing 3% of input and 3% of flow-through are then mixed with 4x sample loading buffer, boiled for 3 min at 95°, and subjected to Western blot analysis, as has been described in Section 2.4. 

Alternatively, GFP can be visualized using rabbit polyclonal antibody raised against GFP purified directly from A. victoria. This anti-GFP antibody facilitates the detection of native GFP, recombinant GFP, and GFP-fusion proteins both by immunofluorescence and brightfield microscopy, as well as by western blot analysis and immunoprecipitation. Direct anti-GFP conjugates made from a complete serum or from an IgG fraction are available from Invitrogen (http //www.invitrogen.com/ site/us/en/home.html). Additional options for your research offered by Invitrogen include two mouse monoclonal antibodies and a chicken IgY fraction. 

Fig. 8 SDS-PAGE (a) and Western blot (b) analysis of the purified hydrogenosomal fractions isolated from the metronidazole-susceptible T. vaginalis strain TV 10-02 (P) and its metronidazole-resistant derivatives MR-3, MR-5, MR-30, MR-50, and MR-100 displaying the aerobic (3), early anaerobic (5), advanced anaerobic (30, 50), and fully developed anaerobic resistance (100) to metronidazole. Numbers in the designation of MR strains indicate the concentrations of metronidazole in ixg/ml at which the organisms multiply in culture. About 10 pg protein was loaded per line. PFOR pyruvate ferredoxin oxidoreduc-tase, a-STK a subunit of succinate thiokinase (hydrogenosomal enzyme not involved in metronidazole resistance used as control), Fdx ferredoxin. From Rasoloson et al. (2002) by courtesy of the Society of General Microbiology...

In vitro. The activity or absolute level of enzymes such as cytochrome P-450 and glucuronosyl transferase can be measured in cells, tissue fractions, or subcellular fractions (e.g., microsomes) and compared with those from control animals. The activity is measured by using a particular substrate for each of the isoforms of the enzyme (e.g., cytochrome P-450 or UDPGT) of interest. The total level of cytochrome P-450 could be determined by spectrophotometry using standard methods (e.g., carbon monoxide binding and difference spectra). Alternatively, the level of protein can be determined by gel electrophoresis and Western blotting, and this would allow the separation of different isoforms. 

In order to investigate whether tomatinases from F. oxysporum and F. solani share similar molecular characteristics, F. solani tomatinase was partially purified. Comparative SDS-PAGE analysis of the protein fractions with and without tomatinase activity showed the presence of a 32.5 kDa band in all positive fractions, while this band was absent in fractions without tomatinase activity The apparent molecular mass of tomatinase of F. solani differs from that of F. oxysporum (50 kDa), S. lycopersici (110 kDa) [33], and Botrytis cinerea (70 kDa)[36]. The F. solani tomatinase presents a very low activity compared with F. oxysporum enzyme [35, 89]. Western blot analysis showed that the two enzymes also differ in their immunological characteristics since the polyclonal antibody against tomatinase of F. oxysporum f. sp. lycopersici did not recognize the tomatinase from F. solani. These results suggest that the enzyme from F. solani is a novel tomatinase species. 

Western blotting has become an important, modern technique for analysis and characterization of proteins. The procedure consists of, first, the electrophoretic transfer (blotting) of proteins from polyacrylamide gels to synthetic membranes. The transferred blots are then probed using immunological detection methods to identify proteins of specific structure and/or function. In this experiment, bovine serum will be fractionated by SDS-PAGE and the proteins blotted onto a nitrocellulose membrane. Serum glycoproteins will be identified by their specific interaction with the lectin concanavalin A. 

B 6. A protein mixture can be fractionated either by native PAGE or by denaturing, SDS-PAGE, before Western blotting. What factors would determine your choice of electrophoresis method ... 

Western blots of microsomal fractions from transfected HEK-293 cells with the PLN-L39stop mutant, indicated that the PLN-L39stop protein could not be detected. In addition, confocal microscopy in HEK-293 cells transfected with PLN-L39stop revealed detectable immunoreactive protein signals in a small percent of cells and the PLN-mutant was mainly localized to the cell membrane, compared with PLN-WT, which localized to the endoplasmic reticulum. Consistent with these findings, human PLN-L39stop homozygous ventricles had no detectable PLN. 

A defining characteristic of mitochondrial and hydrogensomal protein import is the proteolytic removal of amino-terminal targeting presequences upon organelle import. In Giardia, two IscU bands have been observed by western blotting in the organelle fraction that differ in size by about 2 kDa, a... 

In this experiment, you will identify the C-terminal domain of glutathione-S-transferase (GST) from Schistosomajaponicum (purified from an Escherichia coli cell extract in Experiment 10) through the use of a Western blot. After the proteins present in the crude cell extract, unbound fraction, and the fraction eluted from the glutathione Sepharose resin are separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the proteins will be electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane (Fig. 18-1). Although membranes composed of nitrocellulose or nylon can also be used for Western blotting, PVDF membranes are particularly effec-... 

Using the notch made on the film, align it with the notch on the PVDF membrane. Based on the position of the prestained molecular weight protein standards present on the PVDF membrane, determine the molecular weight of the protein band(s) present on the film. Do you believe that it is GST Explain. Are there any other protein bands besides GST that are present on the film (in the crude cell lysate and unbound fraction lanes) If so, explain what this means. Propose how you could alter the conditions of the experiment to prevent these other protein bands from cross-reacting with the antibodies used in the Western blot. 

After fermentation, the yeast cells were harvested and broken mechanically in a bead mill, and differential centrifugation was used to partition the cellular components into water-soluble and insoluble fractions. SDS-polyacrylamide gel electrophoresis in conjunction with Western blot analysis 36) revealed that the polyphenolic protein aggregated with the insoluble cellular protein. 

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