Last updated 10/93 D. Schafer
1. G-buffer There is a 50X stock of this in -20°C freezer in the hallway. This stock does not contain CaCl2, so it must be added to the buffer after it is diluted to 1X. The concentrations of components at 1X are:
2. Actin must be dialyzed against G-buffer+CaCl2. First determine the amount of actin you will need for the number of assays you intend to do. You need approximately 20 - 40 µg of actin for each assay to be run. If using lyophilized G-150 actin, weigh out 3 times by weight of the amount of actin that you need (+10% to account for losses in preparation) of sucrose stabilized actin (i.e., there is 2/3 sucrose by weight in the lyophilized actin). Dissolve the actin at a concentration of ~1 mg/ml in G-buffer+CaCl2 and dialyze ON against G-buffer. After dialysis, centrifuge sample in microfuge to pellet crud, and determine the concentration by measuring the A290 (the extinction coefficient is 0.63 ml/mg.cm). Polymerize the actin at ~1.0 mg/ml by addition of 1/9 volume of 10X MKEI, grade I). Incubate at RT 15-30 min. Dilute actin solution to 0.5 mg/ml. Any leftover actin can be stored at 4°C for up to 2 weeks or until it smells, whichever is longer.
3. 10X MKEI buffer: final conc. (10X) amount of stock (to prepare 50 ml)
Concen in 10X Amount of Stock Concen of Stock
1M KCl 2.5 ml 4M KCl
20 mM MgCl2 1 ml 1M MgCl2
10 mM EGTA 5 ml 0.1M EGTA
200 mM imidazole 10 ml 1M imidazole, pH 7.0 [GRADE I]
Add H2O to final volume = 50 ml
1. Each batch of actin must be tested to determine the appropriate amount to use for the assays. This is done by polymerizing varying concentrations of actin alone and determining the velocity of the falling ball. Want to get a rate of ~0.75-1.0cm/sec using the middle incline on the falling ball assay stand (angle @~50°). This is approximately 8-9 seconds total for the ball to go the entire range.
2. To determine the optimal actin concentration, prepare 200 ml aliquots of actin at concentrations of 0.1 mg/ml to 0.30 mg/ml in 1X MKEI buffer. Use 10mm x 75mm disposable glass tubes to prepare the samples.
3. After each actin addition, vortex each sample uniformly for 5-10 seconds and immediately aspirate into capillary tube and seal bottom. Insert into a RT water bath (13x100mm tubes filled with water) and incubate 10-30 min. Time the preparation of samples so that all the samples get incubated for the same amount of time before adding the falling ball.
4. After the incubation period, assemble the capillary onto the falling ball stand (middle incline) and carefully insert the ball. The ball will sit on the meniscus, and should not fall down into the solution. Use the magnet to start the ball falling through the solution and time with stopwatch for a defined distance (usually 8cm is convenient). Smaller distances are OK, just divide to make sec per cm.
If you accidentally let the ball fall down the tube before you are ready, you cannot tip the tube, bring the ball back and do it again. When the ball rolls through the solution, it breaks and removes a lot of the actin. You can of course estimate how fast the ball was going.
5. Use the actin concentration that gives ~1.0 sec/cm.
Prepare samples with an aliquot of test sample and actin in a final volume of 200ml with 1X MKEI buffer and actin at the concentration determined to be optimal in the test above. Set up the assays so that all tubes are incubated at RT for the same amount of time before testing them with the falling ball.