Optimized Analysis of Proteins from Xenopus Oocytes and Embryos by Immunoblotting

Over the course of an animal's lifetime, sulfate decisions occur continually, that allow for normal development and the health an adult organism. These decisions depend on specific proteins referred to sulfate regulators. The goal of our research is to find the mechanisms that control the synthesis of these critical regulators.

In addition to providing a detailed outline of immunoblotting and Xenopus oocytes and embryos, our protocol gives insight into challenges common to this model organism, such as antibody procurement. Through in-depth study of the binding and repression mechanisms of Bicaudal C, our research has helped establish a foundation of information by which to compare other translational regulators. To begin processing cells for immunoblotting, add 10 microliters of chilled cell lysis buffer diluted to one-times concentration with double deionized water per embryo or oocyte.

Using a micropestle, homogenize the samples on ice. Place the tubes in a benchtop centrifuge and spin the samples at 5, 000 g at four degrees Celsius for 10 minutes to pellet debris, including yolk and insoluble pigments. Transfer the supernatant to a clean 1.5-milliliter tube, ensuring that the pellet is not disturbed during transfer.

Add an equal volume of two-times Laemmli sample buffer supplemented with 5%weight by volume 2-mercaptoethanol to the collected supernatant. Heat the mixture at 95 to 100 degrees Celsius for 10 minutes to denature the proteins. Now, remove a 4 to 12%bis-tris SDS precast gel from its packaging and peel off the sticker from the bottom of the gel.

Insert the gel into the electrode assembly opposite a buffer dam, and place the entire assembly into a vertical electrophoresis tank. Then, fill both the electrode assembly and the bottom of the tank with Tris-MOPS-SDS running buffer. Gently remove the gel comb and pipette running buffer into the wells to eliminate bubbles and equilibrate the buffer.

Now, load five microliters of pre-stained protein standards into the first well and 10 microliters of each prepared sample into the remaining wells. Place the lid on the electrophoresis tank and connect it to a power supply. Then, set the voltage to 200 volts to initiate electrophoresis.

Stop electrophoresis once the sample-buffer dye front exits the gel. Before electrophoresis completes, begin assembling the transfer sandwich in the transfer clip. Fill a glass baking dish with chilled transfer buffer and submerge all transfer materials in this buffer during assembly.

Place the transfer clip in the dish with the black half lying against the bottom, the white half pointed upward, and the clip opened to the left. Lay one or two fiber sponges flat on the black half of the transfer clip. After cutting two pieces of 0.35-millimeter cellulose chromatography paper to size, place one on top of the sponges.

Once electrophoresis is complete, remove the gel from the electrophoresis tank. Using the gel cassette opening lever, carefully pry apart the gel plates, while ensuring that the gel remains attached to one side. Trim off the wells from the top of the gel using the gel releaser.

Place the gel, still attached to one half of the plastic cassette, onto the filter paper, and remove the remaining cassette half so the gel stays flat on the paper. Next, cut a piece of 0.45-micrometer nitrocellulose membrane to match the gel's dimensions. Remove the membrane from its protective paper, briefly wet it in transfer buffer, and carefully place it on top of the gel.

Then, lay a second piece of filter paper on top of the nitrocellulose membrane. Using a roller, gently but firmly press out all air bubbles. Stack one or two additional fiber sponges on top of the filter paper to complete the sandwich.

Close the transfer cassette and clip it tightly to seal the assembled sandwich. Then, insert the closed transfer sandwich into the transfer core with the clip facing upwards, and ensure the black side of the clip faces the black side of the core. Place the transfer core into the electrophoresis tank.

Add an ice pack and a stir bar to the tank, ensuring the stir bar can rotate freely. Fill the tank with chilled transfer buffer until the apparatus is fully submerged, and secure the lid on top. Connect the apparatus to the power supply, match electrode colors, and set the conditions to 100 volts for one hour with the stir bar rotating.

Once the transfer is complete, carefully open the cassette and disassemble the sandwich. Remove the nitrocellulose membrane and mark the side that was in contact with the gel. Then, place the nitrocellulose membrane in a small container so that it lies flat against the bottom.

Cover the membrane completely with Ponceau stain and rock gently on a rocker for 5 to 10 minutes. After pouring the Ponceau stain, rinse the membrane several times with double deionized water until the excess stain is removed and protein bands become visible in the lanes. Then, perform membrane blocking, antibody incubation, and washing steps as shown.

Once the secondary antibody incubation and washing are complete, the membrane is ready for imaging. In a dark room, turn on the film developer and allow it to warm up. Cut two squares of plastic wrap large enough to fully cover the membrane.

Using forceps, place the nitrocellulose membrane face up onto the first square of plastic wrap. In a 1.5-milliliter tube, mix equal volumes of the enhanced chemiluminescence luminol and enhancer reagents. Then, use approximately one milliliter of the mixture to cover most membranes.

Using the plastic wrap, gently manipulate the membrane to ensure the entire surface is evenly coated and incubate for one minute. Then, remove excess ECL reagent by grasping the membrane with forceps and lightly shaking off the liquid. Place the membrane face up onto the second square of plastic wrap, fold it loosely over the membrane, and tape the wrap securely into an autoradiography cassette.

Next, image the membrane using an autoradiography film in a dark room by following the steps shown. After desired protein visualization is achieved, align the developed film with the glow-in-the-dark markers, and use them as a guide to mark the position of the pre-stained protein standards onto the film. Once imaging is complete, carefully remove the membrane from the plastic wrap and immerse it in TBSTW to wash off residual ECL reagent.

Ponceau staining of the membrane confirmed successful protein transfer. Endogenous B1 protein was not detected in oocyte samples, but was clearly detected in stage seven and stage 10.5 embryo samples at approximately 107 kilodaltons. A smaller 70-kilodalton protein band was detected in injected samples across all stages using the Bicc1 antibody, indicating successful expression of the HA-Bicc1 C-terminal fusion.

The HA antibody detected the same 70-kilodalton band only in injected samples, confirming the specificity of the Bicc1 antibody for the fusion protein. Two high-molecular-weight bands, approximately 250 and 270 kilodaltons, were detected in all samples using the CNOT1 antibody. Expression of the 54-kilodalton DDX6 protein was detected in all samples, but was visibly reduced in stage 10.5 embryos.