Antibodies blotting

Primary antibodies blot excess blocking solution from sections, and incubate for 60 min at room temperature or over night at +4°C with a correspondingly diluted primary antibody. Wash sections in PBS or TBS for 2 x 3 min. 

Primary antibodies blot excess blocking solution from sections and incubate for 60 min at room temperature or overnight at +4°C with a mixture of correspondingly diluted unlabeled primary antibodies raised in two different host species (e.g., mouse and rabbit). When using fluorophore-labeled primary antibodies as in direct immunostaining method (one antibody layer), you may skip step (6) with secondary antibodies for indirect immunostaining method (two antibodies layers). Wash sections in PBS for 3 x 3 min. 

Conjugated antibody Blotted antibody Blotted or free Hapten... 

Fig. 3. In vivo interaction between cytohesin 2 and IPdiFl. Coimmunoprecipitation of FLAG-IPCEFl with GFP-cytohesin 2. COS cells were transfected with the indicated expression vectors. After two days of transfection, cells were lysed and immunoprecipitated (IP) with an anti-GFP antibody. After washing, immunoprecipitates were resolved on SDS-PAGE, blotted onto PVDF membranes, and probed with a monoclonal anti-FLAG antibody (Blot) to detect FLAG-tagged IPCEFl. 5% of the input (cell lysates) was also immunoblotted with anti-GFP and anti-FLAG antibodies to ensure that GFP-cytohesin 2 and FLAG-IPCEFl, respectively, were expressed. Reprinted from Venkateswarlu (2003), with permission.

Most sample components analyzed with electrophoretic techniques are invisible to the naked eye. Thus methods have been developed to visualize and quantify separated compounds. These techniques most commonly involve chemically fixing and then staining the compounds in the gel. Other detection techniques can sometimes yield more information, such as detection using antibodies to specific compounds, which gives positive identification of a sample component either by immunoelectrophoretic or blotting techniques, or enhanced detection by combining two different electrophoresis methods in two-dimensional electrophoretic techniques. 

Probe A molecule used to detect the presence of a specific fragment of DNA or RNA in, for instance, a bacterial colony that is formed from a genetic library or during analysis by blot transfer techniques common probes are cDNA molecules, synthetic oligodeoxynucleotides of defined sequence, or antibodies to specific proteins. 

Western blot A method for transferring protein to a nitrocellulose filter, on which the protein can be detected by a suitable probe (eg, an antibody). 

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. 

B) Proteins were resolved by SDS-PAGE, blotted and PG2 polypeptides detected by reaction with anti-PG2 antibodies. Lane 1, 2 pg purified PGl Lane 2, 1 pg purified PG2 Lane 3, 1 pg purified Psubunit. 

Figure 5, Western blot analyses. The blot was probed using antibodies raised against PL3. The PL3 protein bands corresponded to a molecular mass of 42 kDa.

Products were analysed by SDS-PAGE followed by Western blotting (Burnette 1981). RGase antibody production is described by van der Veen et al. 1991 This method can be used to determine the presence of RGase in different enzyme-preparations and culture-filtrates. 

A. nidulans with selected phages of classes A, B, C, D and E resulted in increased polygalacturonase activity in comparison with the wild type strain. The culture media of pga transformants were further analysed by Western blotting with polyclonal antibodies raised against PGI and in all samples examined cross-reactive bands with molecular masses similar to that found for PGI or PGII were detected. 

Figure 3. E. chrysanthemi cell surface labelling with sulfo-NHS-biotin. After labelling, the proteins were separated by SDS-PAGE, blotted onto nitrocellulose and revealed with PemB-antibodies (A) or with streptavidin-peroxidasc (B). Lane 1 A350 (wild type) lane 2 A837 kdgRy, lane 3 A350/pPME6. An arrowhead indicates the PemB position.

The policlonal antibodies raised against the main band of PG also reacted with the 68 kDa band mentioned above. Total proteins from culture filtrates were obtained from both conditions and analyzed by SDS-PAGE. The gel was subsequently blotted and the filter hibridized with the PG antibodies (Fig-3). The results confirmed the presence of a extracellular PG with similar migration in inducing and non-inducing conditions. 

FIGURE 10.3 Structure of the BHTOH-KLH conjugate utilized for raising polyclonal antibodies. Western blots from 2D gels of cytosolic proteins isolated from mouse lung epithelial cells. The blot on the right is from cells incubated with BHT-QM and the blot on the left is from untreated cells. Immunoreactive proteins were identified by electrospray LC-MS analysis of the tryptic digests. Source From Ref. 39, with permission from the American Chemical Society. 

Vande Woude That was true for both mouse and Xenopus. Even though the protein can be detected by western blot, it is occluded from recognition by antibodies at metaphase II arrest. We speculated that this was somehow uncoupling the interaction with microtubules, which activates some kind of checkpoint. This is the only place that this is seen. If you look at CENPE in somatic cells during M phase, it can always be detected. 

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