Fusion western blot

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. 

Aspergillus oryzae Various fusions between the S3mthetic HRP C gene and CIP at both N- and C-termini. Low Recombinant HRP C detected on Western blots (66). 

Fig. I. Examples of Western blots stained with colloidal gold for total protein followed by immunostaining of individual antigensJ.anes 1—3 Proteins on a Western blot from a cytoplasmic extract of poliovirus-infected HEp-2 cells were stained with colloidal gold. The probing monoclonal antibodies, recognizing the viral proteins VP1 and precursor, VPO and VP2, and VP3, respectively, are detected by peroxidase-coupled rabbit-antimouse antibody (asterisks). Lane 4 Western blot of an E. coli lysate, containing a fusion protein composed of protein A and the poliovirus protein 2B. The fusion protein (arrowhead) is detected on the gold-stained blot by peroxidase-coupled IgG that binds to the protein A moiety.

Xgt-11 Infection of E. coli Strain C600 for Western Blot Analysis of (3-Galactosidase-cDNA Gene Fusion Products... 

Transfer the fusion protein from cell lysates to nitrocellulose by Western blotting. Load each track of the polyacrylamide gel with as much antigen as can be clearly resolved. The amount used has to be determined for the particular gel system used... 

The protocol involves a classical SDS-PAGE (10% polyacrylamide) run, followed by transfer onto a Western blot membrane and immunodetection with an anti-pIII antibody. Nevertheless, special care must be taken during sample preparation, because phages are very stable and difficult to denature. The protocol is similar to typical SDS-PAGE sample preparation, except that / -mer cap toe thanol should be replaced by fresh dithiothreitol (DTT, 5 mM final concentration), and the samples should be boiled in a water bath for at least 15 min. Moreover, because the pIII-fusion protein is a minor component of the virion, a large amount of phages should be loaded onto the gel, typically around 1012 phages per lane. 

Determine the approximate expression level of soluble BCCP fusion by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) together with western blot analyses (18) using an anti-His primary antibody (see Note 5). 

To determine the extent of biotinylation of the BCCP fusion protein, cany out a supershift Western blot assay (again with an anti-His primary antibody) in which equivalent crude lysate samples are pre-incubated with or without streptavidin (0.1 g/mL) (Fig. 3 see Note 6). 

The isolation of an antibody is an extremely useful first step toward understanding the function of the protein to which it binds. scFvs derived from phage antibody libraries have been used in immunofluorescence, immunoprecipitation, fluorescence-activated cell sorting, Western blotting, and inhibition of function studies, both in vivo, in tissue culture cells, and in vitro. In this sense, they can essentially be used in the same way as conventional hybridoma-derived antibodies. They have the advantage, however, that the genes for the variable regions are cloned simultaneously with selection. This allows the fusion of functional elements, such as dimerization domains, effector or detector functions to selected scFvs [38], the re-creation of complete... 

The presence of the enzyme-g3p fusion protein should also be detected by western blot analysis with an anti-g3P and/or an anti-enzyme antibody. With anti-g3p detection, the relative intensities of the fusion and the free g3p proteins should be in qualitative agreement with the displayed level evaluated by the ratio between the enzyme and the phage concentration. 

FIGURE 45.3. The effects of DFP on the HSP 27 and HSP 70 levels in myoblast, myoblasts in fusion, and myotube cultures. Levels of HSP 27 and 70 were determined 24 h after addition of DFP at the 10 M concentration. They were quantitated by Western blot with Chemi Genius Biolmaging System (Syngen, Cambridge, UK). Significant difference (Student t-test, p < 0.05 n — 4) between control and DFP-treated cultures was observed in all determinations except for HSP 70 in myoblasts. 

The t-SNARE SNAP-25 is also expressed in the P-cell and has been localized mainly to the plasma membrane (Sadoul etal., 1995). SNAP-25 was cleaved by treatment of SLO-permeabilized cells with botulinum toxins (BoNT) A or E. The two neurotoxins inhibited Ca "-induced insulin exocytosis but failed to abolish the process completely (Sadoul ef al., 1995). This could be due to the failure of the toxins to cleave SNAP-25 already complexed to other fusion proteins (escaping detection by Western blotting) or to the requirement for SNAP-25 at a penultimate step in insulin exocytosis. 

Although mass spectrometry has made significant strides in sensitivity and proteome coverage, a recent report by Weissman and co-workers indicates that significant challenges remain [72]. With the use of a complete fusion, tandem-affinity-purification (TAP)-tag library for the genome of Saccharomyces cere-visiae, protein expression levels were quantitatively determined with Western blots by chemiluminescent detection. These results were compared to MS-based (MudPIT) results [54,73], and the MS data were strongly biased toward the detection of abundant proteins. For the 75% of the proteome that is represented by proteins present at fewer than 5000 copies per cell, only 8% were... 

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