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Save This Selection. CD44 8E2 Mouse mAb Citations Actin filaments have been labeled with DY phalloidin red. Image Gallery Learn more about how we get our images. To Purchase Size Qty. Related Products.
Type: All. NOTE : Please refer to primary antibody product webpage for recommended antibody dilution. Dilute to 1X with dH 2 O. Nonfat Dry Milk : Biotinylated Protein Ladder Detection Pack : Blotting Membrane and Paper : This protocol has been optimized for nitrocellulose membranes. Pore size 0.
Protein Blotting A general protocol for sample preparation. Treat cells by adding fresh media containing regulator for desired time. Aspirate media from cultures; wash cells with 1X PBS; aspirate. Immediately scrape the cells off the plate and transfer the extract to a microcentrifuge tube. Keep on ice. Sonicate for 10—15 sec to complete cell lysis and shear DNA to reduce sample viscosity.
Microcentrifuge for 5 min. Electrotransfer to nitrocellulose membrane Membrane Blocking and Antibody Incubations NOTE : Volumes are for 10 cm x 10 cm cm 2 of membrane; for different sized membranes, adjust volumes accordingly. Incubate membrane in 25 ml of blocking buffer for 1 hr at room temperature. Wash three times for 5 min each with 15 ml of TBST. Incubate membrane with Anti-mouse IgG, HRP-linked Antibody at and Anti-biotin, HRP-linked Antibody at — to detect biotinylated protein markers in 10 ml of blocking buffer with gentle agitation for 1 hr at room temperature.
Proceed with detection Section D. Mix well. Incubate substrate with membrane for 1 minute, remove excess solution membrane remains wet , wrap in plastic and expose to X-ray film. Immunoprecipitation for Native Proteins This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity utilizing Protein G magnetic separation.
Protein G Magnetic Beads : Magnetic Separation Rack : or ATP 10 mM for kinase assays : To prepare 0. Preparing Cell Lysates Aspirate media. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS. Remove PBS and add 0. Scrape cells off the plate and transfer to microcentrifuge tubes. Sonicate on ice three times for 5 sec each. The supernatant is the cell lysate. Immunoprecipitation Cell Lysate Pre-Clearing Highly Recommended A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein G Magnetic beads.
Briefly vortex the stock tube to resuspend the magnetic beads. Incubate with rotation for 20 minutes at room temperature. Separate the beads from the lysate using a magnetic separation rack, transfer the pre-cleared lysate to a clean tube, and discard the magnetic bead pellet.
Proceed to immunoprecipitation section. Pre-wash magnetic beads see Cell Lysate Pre-Clearing section, steps 1 and 2. Transfer the lysate and antibody immunocomplex solution to the tube containing the pre-washed magnetic bead pellet.
Incubate with rotation for 20 min at room temperature. Pellet beads using magnetic separation rack. Keep on ice between washes. Proceed to analyze by western immunoblotting or kinase activity section D. Sample Analysis Proceed to one of the following specific set of steps. Transfer the supernatant to a new tube. The supernatant is the sample. Analyze sample by western blot see Western Immunoblotting Protocol.
Vortex, then microcentrifuge for 30 sec. Transfer supernatant containing phosphorylated substrate to another tube. Immunofluorescence Immunocytochemistry A.
Solutions and Reagents Achieve higher quality immunofluorescent images using the efficient and cost-effective, pre-made reagents in our Immunofluorescence Application Solutions Kit NOTE : Prepare solutions with reverse osmosis deionized RODI or equivalent grade water.
Adjust pH to 8. Mix well then add 0. NOTE : Formaldehyde is toxic, use only in a fume hood. Allow cells to fix for 15 min at room temperature. Aspirate fixative, rinse three times in 1X PBS for 5 min each. Proceed with Immunostaining Section C. Block specimen in Blocking Buffer for 60 min. While blocking, prepare primary antibody by diluting as indicated on product webpage in Antibody Dilution Buffer.
You can see if you look in the tleap. In order to adjust our structures to the force field and remove any model building artifacts, we first perform a several-step equilibration protocol. Several iterations of minimization and molecular dynamics will be preformed with decreasing restraints.
Note that the other parameters not specified are the same as those in the above two files for minimization and molecular dynamics, respectively. When your simulations have finished, you ought to check the stability and realism of results. This ought to also be used to check the validity and stability of your production runs. Once saved onto local machine, transfer it to you working directory on Herbie, Seawulf, etc. Follow usual protocols to do this.
This script will extract the energy, temperature, pressure and volume and averages thereof from the mdout file. To execute, do the following it may be a good idea to make a separate directory just for this analysis, as many files are created :. Filenames, as per this tutorial ought to be 01min, 02md, 03md, etc.
To analyze the whole equilibration experiment i. Please check the results to be sure it worked properly. There are various ways to coordinate the analysis of these output files.. A Production simulation is the ultimate simulation to be performed. Once the structure is built See tLeap section , minimized and equilibrated See Minimization and equilibration section and only essential restraints retained, production dynamics produce the data used to answer the scientific question at hand.
The previous steps were just preparatory. This is the heart of the whole course. The text after this and on this line will be instructions for sander. This is useful in that if you have to run this script more than once, then something must have gone wrong. Thus, this will simply overwrite the files that are erroneous anyways. Don't use this if you really know what your doing and have a reasonable reason for doing so.
The various variables within the input file will determine the nature of the output: How frequently are the energies printed? How detailed is the information? What types of energies are being printed? See Amber10 manual if you don't know what this is. In this case, we're starting with the final snapshot - single frame frame from the previous equilibration run, 9md.
They can contain subtle information in addition to simply the number of atoms in the file first line and the x, y and z coordinates for each atom which is why you need the topology file to give those x,y,z's meaning - what atom has what coordinates so check the Amber10 manual if you're curious.
Asking Professor Rizzo to explain this flag is a good idea, after reading the Amber10 manual don't waste his time , of course, if it is critical to your work. This is the "Big" file. When your simulation is complete, zip the trajectories:. The trajectories are, in my opinion, the most important component of a MD experiment.
So, read the Amber10 Manual. You could notice that ptraj uses the trajectory files to perform the bulk of the data analysis like RMSD, H-bonding evolution, radius of gyration, pi-stacking, etc. Did everything work properly? Did the simulation run to completion? This flag can be useful in debugging failed jobs The frequency this is done is specified is ntwr in the 10md. A restart file will be written at the end of the simulation - the final snapshot of the simulation will be the restart file if and when the simulation has run successfully to completion.
During the simulation, however, this file is continuously re-written as a fail-safe - say the supercomputer crashes during your 10ns simulation, which has been running for 3 weeks. Well, the restart file is printed every ntwr steps so you could, as the name of the flag implies, simply restart the simulation with minor modification to the input file and above runsander.
Thomas Cheatham. See this website. This page contains a brief list of ptraj functions and their syntax. Commands can be combined with most combinations of other functions to suit the need. A useful and recommended program - merely a text file with functional syntax - to write is:. When writing the above, one depressed 'Enter' on the keyboard, which is 'recorded' by vim. So, when the file is executed, it would be like hitting 'Enter' if you were entering the commands by hand in the shell.
It tells the shell to treat the contents of this here file as if the contents were being typed in the shell by hand. RMSD - root mean-square distance - can be used to measure the distance an object moves relative to a reference object. For example, one could use an RMSD analysis to measure the movement of the alpha-carbon atoms in the active site of a protein, using the experimental structure as the reference structure ptraj will measure the RMSD between each object specified in the ptraj script - see below where ptraj will by default fit the two structures, aligning them as much as possible.
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