Gel overlay

Identify the protein spots as usual, i.e., by staining, autoradiography, gel overlay, or Western blot. In the latter case the gel must be separated from the GelBond foil prior to electrotransfer. For this purpose a film remover (Gorg 2003, Fig. 19) is used The gel is placed on the cylindrical remover with foil down, clamped on an edge, and a thin stainless steel or nylon wire is pulled between foil and gel towards to your body. Cover the gel with the wetted blotting membrane (cf Protocol 2.4.3) and transfer membrane as well as gel to the blotting apparatus. 

Tilt casting stand to pour off butanol and rinse gel surface once with running gel overlay. 

Overlay gel with running gel overlay and allow gel to sit while preparing stacking gel. 

Isom Yes. You can see modulation in the absence of P subunits. My student has recently shown by gel overlay and co-immunoprecipitation that tenascin-R binds directly to the a subunit as well as P (Davis et al 2001). They all bind, but they bind different domains of tenascin R. The P subunits bind similar domains and the a subunit binds a different domain. 

BSA was not included in the renaturation buffer nor was the membrane blocked with 5% BSA or other proteins as described elsewhere (Ferrell and Martin, 1991 Celenza and Carlson, 1991) because this reduced the signal observed from CaM kinase II. Incubation of the membrane with BSA or other proteins may, however, increase the signal from other kinases by providing a substrate. Other procedures have employed incorporation of microtubule-associated protein 2 (MAP-2) into the gel matrix to augment detection of CaM kinase II (Kameshita and Fujisawa, 1989) using the gel overlay method however, MAP-2 is a substrate for multiple kinases, thus reducing the specificity of the assay. 

Otto, J. J. (1983) Detection of vinculin-binding proteins with an I-vinculin gel overlay technique./. Cell Biol. 97, 1283-1287. 

FIGURE ] Calcium binding by psoriasin and other members of the SlOO protein family, (a) gel overlay of a two-dimensional gel blot of total protein from noncultured,... 

Arl2 during purification, or to load the GTPase prior to use in the gel overlay or GAP assays. 

Arl2 is loaded to high specific activity with [a- P]GTP for the gel overlay or the TEC-based Arl2 GAP assay and with [7- P]GTP for the charcoal-based GAP assay. For example, to load Arl2 with radiolabeled GTP for GAP or gel overlay assays, we typically use 0.3 //Ci [ P]GTP... 

Fig. 2. The gel overlay assay can detect high-affinity Arl2 binding partners. As described in the text, proteins from a sample of interest are resolved by SDS-PAGE (1) and electrophoretically transferred to nitrocellulose (2). After renaturation of the transferred proteins and blocking (3), the filter is incubated with pre-loaded Arl2 [a- P]GTP (4). Three quick washes at 4° are followed by autoradiography to reveal the location of proteins that retain the Arl2 [a- P]GTP (5). This cartoon shows binding to BART, a 20-kDa protein.

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