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Analysis of proteins by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting (western blotting) is a vital part of the molecular biologist's toolkit. This technique separates a complex protein mixture by molecular weight and then assays the presence of target proteins using specific antibodies. Immunoblotting has a variety of applications. Examples include use as a targeted approach to study protein-protein interactors or as a control to confirm expression or depletion of protein targets. However, the successful execution of immunoblotting requires complicated, multistep experiments. Protocols must be optimized for each organism, target protein, and application. Therefore, knowledge gaps exist for the use of immunoblots in many models, including the model frog Xenopus laevis. Due to their large size, abundant material for biochemical experiments, and facile handling, X. laevis oocytes and embryos have been vital for studying principles of translational control. However, this species lacks specific protocols for robust and routine immunoblotting. Here, we offer an in-depth protocol for western blotting optimized for samples from multiple Xenopus developmental stages. We then analyze translational regulators across development.
Figure 1: Workflow for Xenopus sample preparation and subsequent immunoblot analysis
Figure 2: Processed embryo extract and equipment for immunoblotting. (A) Centrifuged X. laevis stage VI oocyte extract. Lipids and fat-soluble proteins rise to the top while vitellogenin (yolk) and pigmented debris form a pellet. (B) Equipment for SDS-PAGE to separate proteins by molecular weight. (i) Power supply, (ii) Vertical electrophoresis tank, (iii) Electrophoresis tank lid, (iv) Electrode assembly, (v) Buffer dam, (vi) Precast electrophoresis gel; note the green comb (top) and blue sticker (bottom), each of which must be removed. (C) Equipment for membrane transfer. The vertical electrophoresis tank and lid are reused for transfer after a thorough rinse. (i) Roller to remove bubbles, (ii) Transfer clip, (iii) Small stir bar, (iv) Nitrocellulose membrane in between two protective blue sheets of paper, (v) Cellulose chromatography filter paper, (vi) Transfer sponge cushions, (vii) Glass baking dish for transfer assembly, (viii) Ice pack, (ix) Transfer core. (D) Other equipment. (i) Gel releaser (for trimming gel wells pre-transfer), (ii) Gel cassette opening lever, (iii) Forceps (for handling membrane), (iv) Container (for holding membrane), (v) container lid.
Figure 3: Identifying levels of translational regulatory proteins through immunoblotting in X. laevis. (A) Ponceau staining of the immunoblot to identify total protein from X. laevis stage VI oocytes, stage 7 embryos, and stage 10.5 embryos. Each lane represents 10% of the protein prepared from an extract (0.5 oocyte or embryo). (B) Immunoblot analysis of selected X. laevis translational regulatory proteins in stage VI oocytes, stage 7 embryos, and stage 10.5 embryos. Lanes 2, 4, and 6 correspond to samples microinjected with HA-Bicc1 mRNA prior to analysis, while lanes 1, 3, and 5 serve as negative (uninjected) controls. α-Bicc1 antibody was used to assay expression of endogenous Bicc1 across stages (top panel). The blot was subsequently stripped and re-probed with an antibody to the HA affinity tag as a control for α-Bicc1 antibody specificity (second panel). Blots were stripped and re-probed for additional regulatory proteins using α-Cnot1 (third panel) and α-Ddx6 (fourth panel). α-Gapdh (bottom panel) served as a control for equal protein loading. Red arrowheads mark the location of endogenous Bicc1, while black arrowheads mark the location of exogenously expressed HA-Bicc1 C-term.
