The overall goal of the following experiment is to obtain protein complexes from living drosophila embryos using an in vivo cross-linking approach. This is achieved by first loading thousands of flies into a population cage and collecting early embryos. Next, the proteins are cross-linked in vivo with formaldehyde, which stabilizes their internal protein complexes.
Then protein extracts are prepared from the cross-linked embryos so that the protein complexes can be immuno precipitated. The results identify the isolated protein complexes by Western blot analysis. The main advantage of this technique of existing methods, like traditional isolation of protein complexes with in vitro approaches is that this in vivo approach allows us to capture transient but functionally significant protein protein interactions, which might not be detected with traditional in vitro methods.
Demonstrating the technique will be Dr.Min Gao, a postdoc from my laboratory. First mix water and fly agar with a stir bar. Autoclave this mix during the autoclave run.
Mix sucrose into apple juice and heat this to 70 degrees Celsius on a plate. Later, combine the mixtures and allow them to cool for 15 minutes before the agar solidifies. Make fresh methyl four hydroxy benzoate in ethanol.
This is the preservative. Add it to the solidifying mixture and stir it in for five minutes. Then pour the mixture into 300 square centimeter plastic or styrofoam plates without forming bubbles.
Store the plates at four degrees Celsius and use them within a week. Have a population cage of 40 to 50, 000 flies prepared for which to collect embryos. Attach two fresh cornmeal molasses filled plates with about four grams of baker's yeast sprinkled on the surface to the population gauge.
Allow the flies to live on these plates for 48 hours at 25 degrees Celsius. After two days, sprinkle about a gram of yeast onto two apple juice agar plates, and replace the plates in the population cage with these plates. After an hour, replace the plates in the cage with two more of the same type of plate.
The two collection plates that the flies used for an hour will be loaded with embryos. Suspend the embryos and water on the plate and use a brush to help dislodge them. Then pour them through a two layer sieve.
The top layer is an 850 micron steel mesh, and the bottom layer is a 75 micron steel mesh. The embryos will collect on the finer mesh, separated from the coarse debris using a paintbrush. Transfer the embryos to a fine nylon mesh basket made from a 50 milliliter conical tube top.
Rinse the embryos in water and dry them in the mesh against a paper towel. Next, immerse the embryos in a 50%bleach solution for three minutes. Swirl them in the bleach, then thoroughly rinse them off with water and blot them dry.
The embryos are next immersed in isopropanol for about 15 seconds. With swirling, this breaks up the embryo clumps, and it shouldn't take longer than 30 seconds. Blot the embryos dry and then transfer them to heptane solution for five minutes.
Frequently pipette the heptane over embryos to keep the embryos from clumping, blot the embryos dry and rinse them in PBST. After rinsing the embryos for three minutes, transfer them on the mesh to a 15 milliliter tube with 10 milliliters of PBST. Remove the mesh leaving the embryos in solution and watch between 100 and 300 microliters of embryos.
Settle to the bottom, aspirate the PBST and proceed immediately with cross-linking to cross-link the embryos. First, add 10 milliliters of freshly prepared 0.2%formaldehyde in PBST to the embryos. Set them to incubate with gentle agitation at 25 degrees Celsius for 10 minutes.
After 10 minutes of reacting, allow the embryos to sink and remove the solution. Then quickly replace the solution with 10 milliliters of point 25 molar glycine in PBST to quench the reaction. Set the tube to shake for another five minutes After the quenching, remove the solution and rinse the embryos three times using 10 milliliters of PBST For each rinse.
Remove the rest of the PBST by pipette aspiration and store the embryos at minus 80 degrees Celsius until they are needed. Using a down homogenizer and two milliliters of lysis buffer homogenize 500 microliters of cross-linked embryos. This takes about 15 strokes with a tight pestle.
Transfer the lysate to a micro centrifuge tube and set it to rock gently for 15 minutes. Then centrifuge the lysate at 16, 000 GS for five minutes. Transfer the supernatant to a 15 milliliter tube.
The pellet can be disposed of. Now to the supernatant, add 40 microliters of the anti ha agros beet slurry. Set this rocking for about 2.5 hours after the bead conjugation is complete.
Pellet the beads at 2, 500 Gs for five minutes. Leave about 250 microliters of supernatant in the tube and resuspend the beads therein. Transfer the suspended beads to a 10 micron filter on a spin column.
Remove the solution with a ten second spin at 12, 000 Gs.Wash the beads on the column with 200 microliters of wash buffer and another spin down. Do this a total of three times. Then add 40 microliters of elucian buffer to the beads and incubate them at 37 degrees Celsius for 15 minutes.
Gently shake the tube every five minutes during this period to collect the ouit. Spin the column down once more at 12, 000 GS for 10 seconds. The effectiveness of the cross-linking and of the purification of cross-linked two protein complexes was analyzed by SDS page on a three to 7%step gel followed by a western blot embryos expressing HA tag, GFP protein were used to monitor the extent of the cross-linking and test different formaldehyde concentrations for the protocol.
Most of the GFP in the treated embryos did not cross-link, so this does not happen. Randomly cross-linked two complex was compared to UN cross-linked control. Two protein that did not cross-link is seen at a 285 kilodalton band that migrates freely.
Cross-linked two is heavier and migrates less in crude protein lysate. About half of the two protein was cross-linked with its in vivo. Interacting partners elucian with HA peptide by comparison had both complex and free tube protein in the elution fraction, after which washing the beads.
With SDS removed mostly free tube with minimal tube complex. Following this procedure, other methods like mass spectrometry can be performed in order to answer additional questions regarding methods such as the identification of protein components in the isolated complexes.
