The overall goal of this procedure is to purify the in vivo interacting proteins of a gene of interest from the fruit fly adult brain. This is accomplished by first generating the transgenic flies that express the tap tagged bait protein and collecting the adult heads from these flies. The second step is to prepare the supernatant from the fly head lysate for the tap procedure, and then perform the sequential immunoglobin G purification, TEV cleavage and cal modlin purification.
Next five evolution collected from the final step of the tap procedure together with the total brain lysate and the protein complex. After TEV cleavage are resolved by SDS page and visualized by silver staining. Ultimately, the second elution is typically subjected to mass spectrometry for protein identification.
The main advantage of this technique over existing mes like TAL hybrid and core IP experiments, is that the protein protein interactions detected by tap take place under near physiological conditions, which helps preserve the specificity of those interactions. Two rounds of purification steps enable the enrichment of the specific findings and reduce false positive rate. Demonstrating the procedure will be bway through a postdoc from my laboratory.
Prior to starting this procedure, expand the neuronal GAL four UAS tap transgene stock in bottles and flip the bottles every three days until 250 bottles have been used for collection. Collect one to three day old adult flies into a 50 milliliter conical tube and place the tube in liquid nitrogen immediately to deep freeze the flies. Following this store, the flies in a minus 80 degrees Celsius freezer.
Remove two pre chilled USA standard test sieves and a mortar and pestle from the minus 80 degrees Celsius freezer and place them inside a large ice bucket filled with powdered dry ice. Then stack the number 25 sieve on the top of the number 40 sieve in the bucket. After removing the frozen flies from the freezer, place them in liquid nitrogen for approximately 10 minutes.
When finished, shake the tube vigorously to break the heads, legs and wings from the bodies. Next, pour the mixture onto the top sieve and shake the sieves vigorously while holding them together. When finished, separate the two sieves and carefully transfer the fly heads from the bottom sieve to the cold mortar on top of dry ice in an ice bucket, grind the heads with the mortar and pestle to powdery particles and transfer the powder to a pre chilled and pre weighed 15 milliliter glass down tissue grinder.
Then measure the weight of the grinder with the head sample and calculate how much the head sample weighs with the glass grinder on ice. Add 15 milliliters of previously prepared ice cold homogenization buffer to the powder and stroke with the large clearance pestle until the pestle moves up and down the grinder with ease. Following this, transfer the homogenate to a high speed centrifuge tube and spin it for 20 minutes at about 50, 000 times gravity and four degrees Celsius.
When finished, transfer the supernatant to a new high speed centrifuge tube. After repeating the centrifugation, transfer the supernatant to an ultra centrifuge tube and spin it for 40 minutes at about 250, 000 times gravity at four degrees Celsius. To further clear the supernat while the sample is being centrifuged, transfer 400 microliters of immunoglobin g Sphero beads to a 15 milliliter falcon tube.
After adding 10 milliliters of cold immunoglobin G washing buffer, rock the tube gently for two minutes. When finished, spin the beads down at 1000 times gravity for another two minutes. After repeating this process two more times, remove the buffer, leaving only the beads in the tube following centrifugation.
Carefully transfer approximately 15 milliliters of the cleared supernatant to the tube containing the immunoglobin G beads. Incubate the beads and the brain lysate Mix at four degrees Celsius on a mutator for two hours In a cold room, set up a clean and empty 15 milliliter micro column. Load the immunoglobin G bead mixture by steadily pouring it into the column.
After the brain lysate has alluded from the settled immunoglobin G column, wash it thoroughly with 10 milliliters of cold immunoglobin G washing buffer. Repeat the wash twice, making sure not to let the beads dry. Following the third wash, wash the column with 10 milliliters of TEV cleavage buffer right before the last drop of TEV buffer Elutes from the column.
Close the bottom of the column with a cap to block the flow. Then add 1.3 milliliters of TEV buffer containing 130 units of TEV enzyme to the column and close the top with a cap. Rotate the column at 18 degrees Celsius for two hours to allow the TEV enzyme to cleave the peptide at the TEV site and release the protein complex while the immunoglobin beads are being incubated with the TEV enzyme.
Place 200 microliters of Cal Modlin beads in a 15 milliliter Falcon tube and wash them three times with 10 milliliters of cold cal modlin binding buffer For each wash. Rock the tube for two minutes on a mutator and then spin down the beads at 1000 times gravity for two minutes. At the end of the third wash, remove the buffer from the tube.
Once the TEV incubation is complete, set the Immunoglobin G column in the cold room and allow the beads to settle for 10 minutes. Remove the top and bottom caps and collect the 1.3 milliliter TEV cleavage product in a 15 milliliter falcon tube. After allowing the buffer to drain completely, add an additional 200 microliters of TEV buffer to the column and collect the EENT in the same tube.
Next, add 4.5 milliliters of Cal Modlin binding buffer and 4.5 microliters of one molar calcium chloride to the 1.5 milliliter TEV cleavage product tube. Transfer the six milliliter mixture to the tube containing the cal modlin beads. After rotating the tube on a mutator at four degrees Celsius for one hour, set up another clean and empty 10 milliliter micro column in the cold room.
Load the cal modlin bead mixture into the column and allow it to drain by gravity. When all the solution has alluded through the settled Cal Modlin column, carefully wash the column twice with 10 milliliters of cold cal Modlin binding buffer. Immediately after washing gently add 200 microliters of cold cal modlin elution buffer to the column and collect the EIT with a marked 1.5 milliliter einor tube.
Repeat this step four more times to collect a total of five fractions. Following this, analyze the protein contents of each fraction by SDS page. Store the remaining EIT at minus 80 degrees Celsius for further analysis.
Highwire interacting protein and its vertebrate and invertebrate homologs are huge ubiquitin ligase that regulate the development and repair of the nervous system. Work done in worm, fly and mouse led to the current working model that highwire functions as an E three ligase and as a scaffolding protein to facilitate formation of a multi subunit ubiquitination complex, which regulates time and cell type specific neuronal functions. Through the combination of interacting with different co-factors and targeting different ubiquitin substrates.
To identify the highwire associated ubiquitination complex and end terminal tag to UAS tap highwire transgene that is fully functional. In rescuing the highwire mutant phenotype was generated about 10 grams of adult fly heads that expressed tap only or tap highwire Transgenic proteins were collected and subjected to tap procedures side by side as described above. Final EITs from both purifications were analyzed by SDS page and then silver staining mass spectrometry identified a list of proteins in the tap highwire sample only including drosophila FSN and reone.
Subsequent genetic and biochemical analyses revealed that HIGHWIRE and DFSN work together as an SCF, like E three ubiquitin ligase complex to regulate synaptic structure and function, and reone associates with highwire in vivo and restrain synaptic overgrowth. This function of reone is at least partially achieved by its ability to promote highwire protein stability via protecting highwire from autophagic degradation, which reveals a novel mechanism that selectively controls highwire protein abundance during synaptic development. Once mastered, this technique can be done in two to three days if it is performed properly.
While attempting this procedure, it's important to remember to generate the right form of detect transgenes, identify the transform that expresses the transgene at the appropriate level, and lastly, and most importantly, fund the right rous buffer conditions that not only solubilize the base protein, but also preserve the InVivo intact complexes at a relatively stable state. After watching this video, you should have a very good understanding of how to connect doubt, fly has, and perform a tandem affinity purification experiment to purify injecting proteins of your favorite genes in neurons.
