mini gel blot transfer apparatus  (Bio-Rad)

Journal: Methods in Molecular Biology (Clifton, N.j.)

Article Title: Detection and Characterization of Receptor Interactions with PDZ Domains

doi: 10.1007/978-1-61779-160-4_21

Figure Lengend Snippet: Reverse blot overlays and receptor–PDZ affinity estimations. (a) Representative data shown for reverse blot overlay experiments. Briefly, 2 μg of GST–receptor–CT are separated by SDS-PAGE, transferred to nitrocellulose and then cut into strips. Membranes are then overlaid with increasing concentrations of an S-tagged PDZ domain that was identified as a positive hit in the original screen of the PDZ array. Importantly, no binding is seen for overlay of GST alone (data not shown). (b) After converting the immunoreactive bands in the overlay experiments into OD values, the maximum OD value can be identified as that value does not change between two increasing concentrations of S-tagged PDZ domain overlay. The remaining OD values are converted into a percentage of the maximal OD value and plotted onto a dose–response graph, in which the concentration of the PDZ domain is on a logarithmic scale. The curve can then be used to estimate the KD of the receptor/PDZ domain interaction, or the concentration of PDZ domain required for 50% of maximum binding (dashed line).

Article Snippet: Reverse Blot Overlays SDS-PAGE mini-gel apparatus (Invitrogen).

Techniques: SDS Page, Binding Assay, Concentration Assay

Journal: Methods in Molecular Biology (Clifton, N.j.)

Article Title: Detection and Characterization of Receptor Interactions with PDZ Domains

doi: 10.1007/978-1-61779-160-4_21

Figure Lengend Snippet: Immunoprecipitation experiments to validate receptor/PDZ interactions in a cellular context. (a) Schematic diagram illustrating the immunoprecipitation (IP) of a FLAG-tagged receptor using FLAG agarose beads. Solublized cell lysates containing FLAG-tagged receptor and HA-tagged PDZ scaffold are incubated with FLAG-conjugated agarose beads. The FLAG antibody (triangle) that is covalently attached to the beads (circle) immunoprecipitates the FLAG-tagged receptor (gray shape), while the noninteracting protein (pentagon) does not associate. The HA-tagged PDZ scaffold (L-shape) also binds to the FLAG-tagged receptor and is co-immunoprecipitated by the beads. After the incubation, the beads are incubated with 2× Sample Buffer and the receptor and the PDZ scaffolds are eluted. (b) Representative data showing a successful immunoprecipitation (IP) of a FLAG-tagged receptor and the specific co-immunoprecipitation (co-IP) of a HA-tagged PDZ scaffold. Samples from the membrane, soluble lysate, anti-FLAG IP fractions are run on an SDS-PAGE gel, transferred to nitrocellulose, and blotted with an antibody corresponding to the receptor (left blot) and the HA-tag on the PDZ scaffold (right blot). An efficient solubilization of the receptor from the membrane is shown (lane D vs. lane B), and incubation of this soluble lysate with FLAG beads results in a robust immunoprecipitation of the FLAG-tagged receptor (lane F). Likewise, the HA-PDZ scaffold is solubilized efficiently (right blot, lanes I and J vs. G and H, respectively) and a band corresponding to the predicted molecular weight of the HA-PDZ scaffold is only seen in the lane in which the receptor was immunoprecipitated (right blot, lane L). As a negative control, the FLAG beads do not pull-down the HA-PDZ scaffold when the receptor is not co-transfected (right blot, lane K).

Article Snippet: Reverse Blot Overlays SDS-PAGE mini-gel apparatus (Invitrogen).

Techniques: Immunoprecipitation, Incubation, Co-Immunoprecipitation Assay, SDS Page, Molecular Weight, Negative Control, Transfection