中等规模的制备

The overall goal of this experiment, is to provide a straightforward and inexpensive approach to collecting Drosophilia Embryos at medium scale. And preparing protein extracts that can be used in downstream Proteomic applications, such as affinity purification mass spectrometry. This method can help answer key questions in the fields of cell signaling and developmental biology.

For example, how specific protein, protein interactions carry out cell communication during organism development. The main advantage of this technique, is that it's an easy and inexpensive method to generate a Drosophila embryo Lysis. Which can be used for downstream applications like protein affinity purification.

Traditionally, researchers collected Drosophila embryos in very large population cages. But we present a user friendly procedure to collect embryos in medium sized cages that can be easily set up in any lab. After preparing five liter containers and cutting 25 by 25 centimeter squares of nylon mesh, according to the text protocol, place the mesh over the container and press down with the lid.

Ensure the mesh is tight and the lid is not tilted. The cage is now ready to be populated with flies. Following the preparation of one liter plastic jars and 18 by 18 centimeter nylon mesh squares, place the mesh over the top rim of the jar and screw on the lid to tighten.

Make sure the mesh is tight and the lid is not tilted. Amplify the transgenic fly lines carry the typed protein of interest. And transfer the bottles every two days until 25 to 30 bottles are accumulated for each fly line.

Amplify a control stock in a similar manner. Next, warm apple juice plates to room temperature. Then using a gloved finger, spread some wet yeast paste in the center of the plates.

Making an approximately six centimeter circle for a 15 centimeter plate. And a four centimeter circle for a 10 centimeter plate. After anesthetizing the flies, and transferring them into the prepared cages with the mesh facing down, cover the cages with apple juice plates.

Allow the flies to wake up. Then turn the cages over and incubate the flies at room temperature for two to three days. Change the plates twice per day, by turning the cage upside down, and while holding the plate to the cage, peel the tape from the plate.

Gently tap the whole cage on the bench and quickly swap the old plate for a new one, trying not to allow the flies to escape. Allow flies to lay embryos on fresh apple juice plates overnight, or for any other desired length of time. The following morning, mark and weigh mesh containers for embryo collection.

One for each fly line used. Prepare 1x Lysis Buffer in a 50 milliliter tube using previously made reagents, by adding 40 milliliters of water to 10 milliliters of 5x concentrated Lysis Buffer in a 50 milliliter tube. Add 50 microliters of one molar DTT at a final concentration of one mili molar.

Mix well and divide the solution between two 50 milliliter tubes. To one of the tubes add one protease inhibitor tablet. And place the tube on a rotator at four degrees Celsius for 30 minutes.

While the tablet is dissolving, start collecting the embryos by marking the apple juice plates on the cages, with the genotypes of fly lines used. And swap the plates for fresh ones. Add enough water to cover each apple juice plate.

Then with a soft paintbrush, gently dislodge the embryos from the plate. Pour the embryos into the previously weighed and marked mesh container and drain the excess water. Then briefly blot the mesh on paper towels to remove the excess water.

To dechorionate the embryos, half fill a six centimeter Petri dish with 50%bleach. Then prepare two larger Petri dishes with water. Immerse the mesh container with embryos, in 50%bleach for 90 seconds.

Gently tap the mesh during incubation a couple of times, to suspend the embryos in the bleach solution. At the end of the incubation, use water to wash the mesh container in each of the two dishes, for about 10 seconds. Then use 18.2 megohm centimeter water in a squirt bottle, to thoroughly rinse the mesh container, including the sides and the outside.

No smell of bleach should be detected after a thorough wash. Blot the mesh container on paper towels. Then weigh the mesh container, with the embryos and subtract the weight of the empty container.

Record the resulting weight of the embryos. To prepare embryo extract, add one milliliter of Lysis Buffer with protease inhibitors, to a Dounce glass homogenizer and keep it on ice. Then transfer the embryos from the mesh container into the homogenizer.

Use one milliliter of Lysis Buffer to wash off the remaining embryos on the mesh. And add the embryos and buffer to the material in the homogenizer. Then let the embryos settle down to the bottom of the homogenizer and carefully aspirate the supernatant.

Next, add Lysis Buffer with protease inhibitors to the homogenizer, aiming for five to six milliliters of Lysis Buffer per gram of embryos. Then insert a loose fitting pestle to homogenize the embryos with four to six strokes. During the first strokes, there will be more resistance.

Try to move the pestle in a continuous motion, to avoid splashing the solution. Then switch to a tight fitting pestle. And homogenize the embryos with eight to 10 strokes.

Incubate the homogenid on ice for 20 minutes. Then transfer the homogenid into micro centrifuge tubes. And centrifuge at 13, 000 time G and four degrees Celsius for 15 minutes.

While avoiding the pellets in the very top lipid layer, transfer the supernatants into fresh micro centrifuge tubes on ice. After repeating the centrification use a one milliliter tip to carefully collect the supernatants. And transfer them into chilled, 10 milliliter syringes, with 045 micrometer filters attached.

Push all of the solution into labeled two milliliter micro centrifuge tubes on ice. Snap freeze the samples in liquid Nitrogen and store them at negative 80 degrees Celsius. Or use immediately for protein purification experiments.

Using the protocol described in this video, Lysates were prepared from Drosophila embryos ubiquitously expressing the EGFP-ERK protein, under control of the arm-odillo promoter. The expression of protein was confirmed by Western Blotting for total ERK protein to simultaneously detect the levels of endogenous and transgenic EGFP-ERK, migrating at 42 kilodaltons and 69 kilodaltons respectively. A single band, corresponding to untagged endogenous ERK was detected in the yellow white control line, confirming a successful extraction of ERK and EGFP-ERK from embryos.

To test whether the extraction protocol is suitable for subsequent purification of tagged protein, extracts from the yellow white, and arm-EGFP-ERK flies, were subjected to affinity purification, using GFP Agarose Beads. Silver staining of samples run on a gradient SDS-PAGE, show successful purification of the bait protein EGFP-ERK. Besides EGFP-ERK, the experimental lane contained additional bands, not observed in the control sample, suggesting that the purification recovered potential ERK interacting proteins.

Once mastered, the embryo collection and the extraction steps, can be done in approximately two hours. While attempting this procedure it's important to remember to perform the extraction steps on ice. And to use protease inhibitors to minimize protein degradation.

After watching this video, you should have a good understanding of how to set up Drosophila population cages at medium scale. And make whole cell protein extracts from embryos.