In the cell. The Acton Cytoskeleton is a dynamic structure, and its remodeling is important in a variety of processes from cell division, cell migration, and physical transport. A wide variety of proteins can bind to the actin cytoskeleton through distinct actin binding modules.
In this video, we demonstrate the procedure of acting co sedimentation, which is an in vitro assay, routinely used to analyze proteins or specific domains of proteins that bind filamentous actin. Hi, I'm Jody Var from Dr.Diane Barber's laboratory at the Department of Cell and Tissue Biology at UCSF. In this video, I'm gonna show you a procedure of how to carry out an actin co sedimentation assay.
This procedure is one method to analyze proteins that specifically bind into filamentous actin and can be used to measure binding affinities. This procedure involves the following steps, preforming actin filaments and preparation of protein to be tested. Incubating protein of interest with filamentous actin, or f actin for short pelleting F actin by ultracentrifugation at a hundred thousand GSDS page and kuci staining to visualize supinate and pellet fractions.
And finally, analysis of the protein co sedimenting with f actin in the palate fraction. So let's get started. In order to prepare actin filaments, it is good practice to ensure that the actin that we are working with is in mono form.
A stock solution of actin at 10 M per mil is removed from the minus 80 freezer and a working dilution 2.4 mgs per mil is made in a buffer containing tris calcium chloride, A TP for monomer stabilization and DTT. The working dilution is placed on ice for one hour after one hour incubation on ice. The Acton solution is centrifuged at 14, 000 RPM for 10 minutes at four degrees.
The resultant sup natant will be the monomeric acton. Now, from these purified acton monomers, filamentous acton can be generated by the addition of potassium chloride, magnesium chloride, and a TP for one hour. At room temperature, the filamentous actin is now ready to incubate with the protein or the protein domain of interest, which we will show you in the next step.
To proceed with the actin co sedimentation assay, we must prepare the protein to be tested. Today I'm using the clan, which has been purified as a GSC fusion protein. The protein is pre-cleared of aggregates by ultracentrifugation and a resulting sup Natin is now ready to use in the assay.
In order to test protein binding to actin, actin must be in excess Here in this case, 10 micrograms of actin is incubated with five micrograms of protein in a buffer containing tris, A TP BT ME capto ethanol ETTA calcium chloride to yield a total volume of 150 microliters for the protein actin incubation, the incubations are carried out directly in ultracentrifuge tubes for one hour at room temperature. Tubes one and two represent controls tube. One contains acting only in buffer tube.
Two contains protein only in buffer tube. Three contains both acting and the CNU of tailn in buffer two four is an additional control for non-specific binding, which contains actin and GST protein alone. After the one hour incubation, our samples are carefully placed into the Beckman rotor.
We then set the ultracentrifuge at a hundred thousand G and run the samples for 20 minutes at 22 degrees C.After this spin, we were able to prep our samples for running on SDS page. Following the 20 minute spin, the samples are carefully removed from the rotor so that the is not disturbed. A mark is made on the side of the tube where the palate is located.
Care is taken to transfer the supinate to einor tubes to which five XSCS sample buffer is added. One XSCS sample buffer is added to the palate remaining in the ultracentrifuge tubes. After petting up and down, the Resus suspended palate is transferred to a fresh eend orph tube.
The samples are then boiled in a heating block for up to 10 minutes. After boiling samples are loaded onto an SDS page gel. Here, I'm using a 10%gel already prepared.
The samples are carefully loaded into the wells in correct order. The gel is run at constant bolts for approximately one hour at room temperature. After the gel has finished running, we stain it with kumasi blue to visualize the proteins.
Kumasi blue solutions should cover the gel completely. The gel is left in a shaker for at least 20 minutes. Then the ESI blue solution is removed and the gel is immersed in detain solution.
The gel is left to detain for approximately one hour on a shaker until bands are seen in a gel. After kumasi staining, the gel can be analyzed to determine the relative amount of protein in the supinate versus the palate fractions. Looking at the gel that repaired in lanes one and two, we can see the snat and pellet fractions of acton alone.
In lanes three and four, we had the snat and pellet fractions of the C teer tailn alone. In lanes five and six, we had the snat and pellet fractions of the acton and C of tail and incubation. In lanes seven and eight, we had the supinate and palate fractions of the actin and GST protein incubation.
As you can see, the CT of tail is only visible in the palate fraction in lane six. The results of this gel indicate a specific binding of the C term, the tail to acton. In this video, you have watched how to carry out an Acton co sedimentation assay.
Acton Co sedimentation assays are an in vitro method to analyze acton binding proteins. When doing this procedure is important to remember these points. To pre-clear your protein of interest to include a negative control of protein alone for every protein actin incubation.
This control will show if any of the pre-cleared protein sediments on its own, that binding to f acting by some proteins can be sensitive to pH temperature or salt concentration in the buffer. So that's it. Thank you for watching and good luck with your experiments.
