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

Journal: Methods (San Diego, Calif.)

Article Title: Reconstitution and Analyses of RNA Interactions with Eukaryotic Translation Initiation Factors and Ribosomal Preinitiation Complexes

doi: 10.1016/j.ymeth.2019.03.024

Figure Lengend Snippet: eIF3 purification from yeast. Lysate from cells overexpressing His-tagged eIF3B were fractionated on Nickel, Phosphocellulose (P11), and Superdex 200 columns to purify the core eIF3 complex. Chromatograms are shown for Nickel and Superdex columns, with A260 and A280 traces shown in red and blue, respectively and percentage buffer B (high imidazole) in orange. 10% SDS-PAGE gels stained with Coomassie are shown. The gel run following P11 (center) shows 1) clarified lysate, 2) MW marker, 3) P11 bound, 4) Ni eluate/P11 load, 5) P11 flowthrough, 6) P11-B200 eluate, 7) P11-B350 eluate. Asterisks mark contaminants that are consistently found in B350/1000 fractions. The larger contaminant is associated with RNase activity and the lower is a similar size to eIF3 ligands eIF5 and eIF2-gamma. The lower gel shows the final purified protein next to MW marker (Precision plus, Biorad: 250, 150, 100, 75, 50, 37, and 25 kDa bands), with the subunits and approximate MWs indicated.

Article Snippet: SDS PAGE mini-gel apparatus (e.g. Biorad #1658000) and power supply Cooled vertical electrophoresis apparatus (e.g. Hoefer #SE260–10A-.75) Refrigerated Circulator for native electrophoresis (e.g. Thermo #HAK1525108) Circulating water bath for biochemical assays (e.g. Fisher #13-874-177) 10K Molecular weight cutoff (MWCO) dialysis cassettes (Thermo #66810) 10K MWCO protein concentrators (Millipore #UFC901024) 0.45 μm syringe filters (Millipore #SLHV033RB) LB Powder (BD #214906) YPD Powder (Teknova #Y5301) Carbenicillin (Goldbio #C-103) *Note: Ampicillin may also be used, but is less stable than Carb, so plates must be made frequently and will give satellite colonies lacking plasmid of interest.

Techniques: Purification, SDS Page, Staining, Marker, Activity Assay

Journal: Methods (San Diego, Calif.)

Article Title: Reconstitution and Analyses of RNA Interactions with Eukaryotic Translation Initiation Factors and Ribosomal Preinitiation Complexes

doi: 10.1016/j.ymeth.2019.03.024

Figure Lengend Snippet: Purification of Intein-Fusions of yeast eIF4B and eIF4G from E.coli. A. eIF4B was expressed as an intein-chitin-binding domain (CBD) fusion protein, and purified by running over a chitin column and eluting by intein cleavage with DTT buffer. Chromatogram shows eluate fractionation over a Heparin column. Gel shows Precision plus marker (M; Biorad) next to two dilutions of purified eIF4B. B. eIF4G fused to the intein-CBD was purified by binding to and washing on chitin resin followed by on-column nuclease digestion with micrococcal nuclease. Further washing was followed by DTT-induced intein-cleavage to elute eIF4G (complexed with eIF4E or without eIF4E). Chromatogram of anion exchange (Q) chromatography of eIF4G•eIF4E chitin eluate, from which full-length eIF4G eluted after degradation products. SDS-PAGE analysis shows separation of eIF4G•eIF4E (1–10) or eIF4G (11–13) from contaminants, with following loading order: M - Marker, 1) Lysate, 2–3) Low Speed supernatant, 4) Chitin Beads, 5) Q load, 6) Q flowthrough, 7–10) fractions from peaks 1 and 2, 11) Chitin Beads, 12) Q load, 13) Q flowthrough, and 14) major eIF4G peak fraction.

Article Snippet: SDS PAGE mini-gel apparatus (e.g. Biorad #1658000) and power supply Cooled vertical electrophoresis apparatus (e.g. Hoefer #SE260–10A-.75) Refrigerated Circulator for native electrophoresis (e.g. Thermo #HAK1525108) Circulating water bath for biochemical assays (e.g. Fisher #13-874-177) 10K Molecular weight cutoff (MWCO) dialysis cassettes (Thermo #66810) 10K MWCO protein concentrators (Millipore #UFC901024) 0.45 μm syringe filters (Millipore #SLHV033RB) LB Powder (BD #214906) YPD Powder (Teknova #Y5301) Carbenicillin (Goldbio #C-103) *Note: Ampicillin may also be used, but is less stable than Carb, so plates must be made frequently and will give satellite colonies lacking plasmid of interest.

Techniques: Purification, Binding Assay, Fractionation, Marker, Chromatography, SDS Page