The overall goal of this procedure is to isolate active full-length kinesin from drosophila embryos for single molecule biophysical studies. The first step of the procedure is to collect embryos and coate them with bleach. Next, the coated embryos are homogenized and the lysates are clarified.
Then microtubules are polymerized in the clarified embryo, lysate and kinesin is stably bound to the microtubules. Finally, the kinesin microtubule complex is sedimented and the kinesin is released from the microtubules. Ultimately, the ability of the kinesin to walk along microtubules can be assessed in a single molecule bead assay using an optical trap.
This protocol is a modification of a protocol developed by bi Saxton for purifying drosophila kinesin. And one of the challenges faced by many beginning biophysicists is the need for functional protein. Physicists are not usually trained in in making proteins, so we develop this protocol to try and make clear and easy how it is that you can purify protein so that biophysicists will have an easy source of of kinesin.
Kinesin is interesting because it's a mechano enzyme, so not only is it a protein, but it walks and it exerts force, and it's a very small nano machine or molecular motor, and we'd like to understand how it works and this protocol will make it possible for people to understand it better. Generally, individuals new to this matter will struggle to get enough embryos for this purification protocol. This can be achieved by carefully maintaining the flies to get 58 laying cups from around 10 grams of embryos, we'll get around 200 micrograms of kinesin at the end of this protocol.
If the flies are not L laying well, you could collect three sets of collection plates, overnight plates, and store at four degrees. Alternatively, you can perform every lysates, every overnight collection store at minus 80 for at most one month, and proceed with further. Proceed further with the protocol as as soon as you have enough BR eight.
The flies light best between two and days old, two to 10 days old and delaying ability decreases after 15 days. To prepare fly cups begin by using a marker to make outlines on the sides of 100 milliliter tricorn beakers using a heated blade cut out holes. Use sticky gorilla tape to cover the holes with nylon mesh.
After amplifying about 50 vials of flies to maximum capacity, flip the amplified flies into an empty plastic vial until they have reached one inch of the vials height. Transfer the flies into fly cups, continuously tapping the cups on a surface to prevent the flies from escaping. Cover each cup with an agar plate containing a yeast paste and incubate the flies at 25 degrees Celsius for 24 to 48 hours.
To stabilize, collect the overnight agar plates from 50 cups and using a clean paintbrush and running water. Wash the contents of the plates into a sieve catcher. Flush the embryos with plenty of water until all the yeast paste is washed away.
Next to dec coate the embryos immerse them in 50%bleach for three minutes. Then use distilled water to rinse the embryos extensively until they lose the bleach odor. Dry the mesh with the embryos by repeatedly placing it on a folded paper towel.
Transfer the embryos to a clean weighing dish and record the weight. Homogenize the embryos by first placing them in a dance homogenizer with 1.5 volumes of ice cold extraction buffer. Perform five strokes with a pestle, then aliquot the homogenate into clean centrifuge tubes.
Centrifuge the homogenate at 15, 000 G for 40 minutes at four degrees Celsius. Carefully collect the clear supernatant liquid without the upper white lipid layer or the lower pellet. Transfer it to clean centrifuge tubes and centrifuge at 50, 000 G for 30 minutes at four degrees Celsius.
Use the supernatant immediately or snap, freeze it and store it at negative 80 degrees Celsius to begin the kinase and binding assay. If frozen, start by thawing the high speed supernatant to room temperature polymerize microtubules by combining one milliliter of supernatant with one microliter of 0.3 molar GTP and two microliters of 10 millimolar Taxol. Agitate gently at room temperature for 20 minutes in a rotating shaker.
Then bind the kinesin to microtubules by adding 2.5 millimolar of the non hydrolyzed A TP analog A-M-P-P-N-P and agitated room temperature for 10 minutes in a rotating shaker sediment, the microtubules in kinesin through an equal volume sucrose cushion by centrif deviation at 23, 000 G for 30 minutes at four degrees Celsius. Wash the pellet with extraction buffer containing 10 micromolar Taxol and 75 millimolar sodium chloride. Repeat the sedimentation with another equal volume sucrose cushion resus.
Suspend the pellet from the salt wash in 5%extraction buffer containing 20 micromolar Taxol 75 millimolar sodium chloride, 10 millimolar magnesium sulfate, and 10 millimolar A TP.Sediment the sample at 120, 000 G for 20 minutes at four degrees Celsius. Then collect the kinesin containing supernat infraction. Perform a centrifugal filtration at 14, 000 G for 10 minutes at four degrees Celsius.
Recover the concentrated sample and using a new filter, repeat the filtration for 30 minutes. Collect the concentrated sample and assay to determine the protein concentration aliquot and snap freeze in liquid nitrogen. Then store it negative 80 degrees Celsius.
This figure shows a silver stained gel of the fractions collected during a kinase and purification lanes one and two are the high speed supernatant and pellet respectively. After clarification, lane's three through six represent the supernatants and pellets respectively after the first and second sucrose cushions. Blaine seven is the pellet after the final sedimentation.
Lane's eight and nine are the purified and final filtered kinase and samples showing the 115 kilodalton kinase and heavy chain. One shown here is a western blot of the purified kinase infraction detected using the kinase kinesin heavy chain antibody. The antibody, A-K-I-N-O one A does not significantly cross-react with other kinesin family members.
The processive of kinesin was evaluated by an in vitro single molecule microtubule binding assay described in more detail in the written protocol. This movie shows a 500 nanometer diameter bead with a single kinesin motor walking along microtubules. The length of the video screen represents 20 microns.
This figure represents the measured distribution of run lengths for single full length purified drosophila kinesin molecules. The exponential fit to the distribution provides the average run length of single kinesin as 1.55 plus or minus 0.1 microns and 1.28 plus or minus 0.12 microns for the filtered and unfiltered samples respectively. Once mastered, this protocol can be finished within seven hours.
Starting from the embryo washing step, the most important final step is to confirm the functionality of the purified cent in our lab. This has been performed using a single molecule B assay involving an optical trap system. In this single molecule experiments, we usually measured the biophysical properties of the purified kinon, such as the motor travel distance velocity, and the force production.
This measured parameter values can give direct evidence of the functionality of kinon. After seeing this protocol, you should have a pretty good idea of how to purify full length drosophila kinesin with the light chains from drosophila embryos, and this should be a very useful process in able enabling you to study kinesin. Kinesin is a motor protein and it's failure is involved in a lot of neurodegeneration, and so a lot of mechanistic studies should help us understand the link between a mechanistic link between disease and motor function.
