What the experts say about colloidal silver and other silverbased antimicrobials. While the FDA uses their legal prerogative to assert that colloidal silver has. Water memory is the purported ability of water to retain a memory of substances previously dissolved in it even after an arbitrary number of serial dilutions. Batimastat BB94 is a potent, broad spectrum matrix metalloprotease MMP inhibitor for MMP1, MMP2, MMP9, MMP7 and MMP3 with IC50 of 3 nM, 4 nM, 4 nM, 6 nM and. Content/images/DilutionDrawing.png' alt='Why Are Serial Dilutions Used' title='Why Are Serial Dilutions Used' />Blog Archive Analyzing gels and western blots with Image. JThe following information is an updated version of a method for using Image. J to analyze western blots from a now deprecated older page. If youre looking for a more comprehensive workflow option for your western blot analyses, please visit my tutorial on using Image Studio Lite, a free software package from LI COR Biosciences. The Image Studio Lite software can be downloaded for free from www. In addition to the functionality described below for Image. J, Image Studio Lite provides additional data management features along with annotation tools for producing publication ready images of your westerns. A pdf copy of this page is available. How To Faster From Filecloud.Io. Image. J http rsb. This tutorial assumes that you have carried your gel or blot through the visualization step, so that you have a digital image of your gel in. If you are scanning x ray film on a flatbed scanner, make sure you use a scanner with the ability to scan transparencies i. EDG2012/p18u01qupppt71r77e6e1qgr123h5_w600_h0.png' alt='Why Are Serial Dilutions Used In Biological Experiments' title='Why Are Serial Dilutions Used In Biological Experiments' />See the references at the end of this tutorial for a discussion of the various ways that you can screw this step up. This also assumes that you didnt overexposed your blot image when you produced it. For an explanation of why overexposure will ruin your results, see this page. The method outlined here uses the Gel Analysis method outlined in the Image. J documentation Gel Analysis. PoMl9jYdl0/UABho-eKJiI/AAAAAAAABhc/RUgE-0gg8NU/s1600/cfu_serial+dilution.jpg' alt='Why Are Serial Dilutions Used In Elisa' title='Why Are Serial Dilutions Used In Elisa' />You may prefer to use it instead of the methods I outline below. There should be very little difference between the results obtained from the various methods. This version of the tutorial was created using Image. J 1. 4. 2q on a Windows 7 6. Open the image file using File Open in Image. J. 2. The gel analysis routine requires the image to be a gray scale image. The simplest method to convert to grayscale is to go to Image Type 8 bit. Your image should look like Figure 1. Figure 1. A fabricated western blot image opened in Image. J. The information along the top of the image indicates that the image is currently in 8 bit mode, using an inverting LUT look up table. The inverting LUT ensures that dark bands will be recorded as higher density values. Choose the Rectangular Selections tool from the Image. J toolbar. Draw a rectangle around the first lane. Image. J assumes that your lanes run vertically so individual bands are horizontal, so your rectangle should be tall and narrow to enclose a single lane. If you draw a rectangle that is short and wide, Image. J will switch to assuming the lanes run horizontally individual bands are vertical, leading to much confusion. Figure 2. The first lane has been selected with the Rectangular Selections tool. After drawing the rectangle over your first lane, press the 1 Command 1 on Mac key Command 1 on Mac or go to Analyze Gels Select First Lane to set the rectangle in place. Why Are Serial Dilutions Used In Microbiology' title='Why Are Serial Dilutions Used In Microbiology' />The 1st lane will now be highlighted and have a 1 in the middle of it. Use your mouse to click and hold in the middle of the rectangle on the 1st lane and drag it over to the next lane. You can also use the arrow keys to move the rectangle, though this is slower. Center the rectangle over the lane left to right, but dont worry about lining it up perfectly on the same vertical axis. Image J will automatically align the rectangle on the same vertical axis as the 1st rectangle in the next step. Press 2 Command 2 on Mac or go to Analyze Gels Select Next Lane to set the rectangle in place over the 2nd lane. A 2 will appear in the lane when the rectangle is placed. Repeat Steps 5 6 for each subsequent lane on the gel, pressing 2 Command 2 on Mac each time to set the rectangle in place Figure 3. Figure 3. Place the rectangle over each lane, pressing 2 each time to set the rectangle in place. After you have set the rectangle in place on the last lane by pressing 2, press 3 Command 3 on Mac, or go to Analyze Gels Plot Lanes to draw a profile plot of each lane. Figure 4. The profile plot from the example western blot. The profile plot represents the relative density of the contents of the rectangle over each lane. The rectangles are arranged top to bottom on the profile plot. In the example western blot image, the peaks in the profile plot Figure 4 correspond to the dark bands in the original image Figure 3. Because there were four lanes selected, there are four sections in the profile plot. Higher peaks represent darker bands. Wider peaks represent bands that cover a wider size range on the original gel. Images of real gels or western blots will always have some background signal, so the peaks dont reach down to the baseline of the profile plot. Figure 5 shows a peak from a real blot where there was some background noise, so the peak appears to float above the baseline of the profile plot. It will be necessary to close off the peak so that we can measure its size. Figure 5. Real western blots often have some background noise, so the peak doesnt reach down to the baseline perfect white. Choose the Straight Line selection tool from the Image. J toolbar Figure 6. For each peak you want to analyze in the profile plot, draw a line across the base of the peak to enclose the peak Figure 5. This step requires some subjective judgment on your part to decide where the peak ends and the background noise begins. Figure 6. Use the straight line tool to close off the base of each peak of interest. Figure 7. The peak from Figure 5 is shown with a line drawn across the base of the peak to enclose the area of the peak. Note that if you have many lanes highlighted, the later lanes will be hidden at the bottom of the profile plot window. To see these lanes, press and hold the space bar, and use the mouse to click and drag the profile plot upwards. When each peak has been closed off at the base with the Straight Line selection tool, select the Wand tool from the Image. J toolbar Figure 8. Figure 8. Choose the Wand tool to highlight each peak of interest on the profile plot. Using the spacebar and mouse, drag the profile plot back down until you are back at the first lane. With the Wand tool, click inside the peak Figure 9. Repeat this for each peak as you go down the profile plot. For each peak that you highlight, measurements should pop up in the Results window that appears. Figure 9. Click inside the 1st peak of interest with the Wand tool. The peak should be highlighted and a measurement should pop up in the Results window. When all of the peaks have been highlighted, go to Analyze Gels Label Peaks. This labels each peak with its size, expressed as a percentage of the total size of all of the highlighted peaks. The values from the Results window Figure 1. Edit Copy All in the Results window. Paste the values into a spreadsheet. Figure 1. 0. The output from selecting peaks in the profile plot and labeling the peaks. In this case, the results are from selecting the upper band in each of the 4 lanes in the example western blot from Figure 1. Note If you accidentally click in the wrong place with the Wand, the program still records that clicked area as a peak, and it will factor into the total area used to calculate the percentage values. Obviously this will skew your results if you click in areas that arent peaks. If you do happen to click in the wrong place, simple go to Analyze Gel Label Peaks to plot the current results, which displays the incorrect values, but more importantly resets the counter for the Results window. Water memory Wikipedia. Water memory is the purported ability of water to retain a memory of substances previously dissolved in it even after an arbitrary number of serial dilutions. It has been claimed to be a mechanism by which homeopathic remedies work, even though they are diluted to the point that no single molecule of the original substance remains. Water memory defies conventional scientific understanding of physical chemistry knowledge and is not accepted by the scientific community. In 1. 98. 8, Jacques Benveniste published a study supporting a water memory effect amid controversy in Nature, accompanied by an editorial by Natures editor John Maddox urging readers to suspend judgement until the results could be replicated. In the years following publication, multiple supervised experiments were run by Benvenistes team, the United States Department of Defense,1BBCs Horizon programme,2 and other researchers, but no team has ever reproduced Benvenistes results in controlled conditions. Benvenistes studyeditBenveniste was a French immunologist who sought to demonstrate the plausibility of homeopathic remedies independently of homeopathic interests in a major scientific journal. To that end, Benveniste and his team at Institut National de la Sant et de la Recherche Mdicale INSERM, French for National Institute of Health and Medical Research diluted a solution of human antibodies in water to such a degree that there was virtually no possibility that a single molecule of the antibody remained in the water solution. Nonetheless, they reported, human basophils responded to the solutions just as though they had encountered the original antibody part of the allergic reaction. The effect was reported only when the solution was shaken violently during dilution. Benveniste stated Its like agitating a car key in the river, going miles downstream, extracting a few drops of water, and then starting ones car with the water. At the time, Benveniste offered no theoretical explanation for the effect, which was later called water memory by a journalist reporting on the study. ImplicationseditWhile Benvenistes study demonstrated a mechanism by which homeopathic remedies could operate, the mechanism defied conventional scientific understandingclarification needed of physical chemistry knowledge. A paper about hydrogen bond dynamics9 is mentioned by some secondary sources1. Publication in NatureeditBenveniste submitted his research to the prominent science journal. Nature for publication. There was concern on the part of Natures editorial oversight board that the material, if published, would lend credibility to homeopathic practitioners even if the effects were not replicable. There was equal concern that the research was simply wrong, given the changes that it would demand of the known laws of physics and chemistry. The editor of Nature, John Maddox, stated that, Our minds were not so much closed as unready to change our whole view of how science is constructed. Rejecting the paper on any objective grounds was deemed unsupportable, as there were no methodological flaws apparent at the time. In the end, a compromise was reached. The paper was published in Nature Vol. June 1. 98. 8,4 but it was accompanied with an editorial by Maddox that noted There are good and particular reasons why prudent people should, for the time being, suspend judgement and described some of the fundamental laws of chemistry and physics which it would violate, if shown to be true. Additionally, Maddox demanded that the experiments be re run under the supervision of a hand picked group of what became known as ghostbusters, including Maddox, famed magician and paranormal researcher James Randi, and Walter W. Stewart, a chemist and freelance debunker at the U. S. National Institutes of Health. Post publication supervised experimentseditUnder supervision of Maddox and his team, Benveniste and his team of researchers followed the original studys procedure and produced results similar to those of the first published data. Maddox, however, noted that during the procedure the experimenters were aware of which test tubes originally contained the antibodies and which did not. Benvenistes team then started a second, blinded experimental series with Maddox and his team in charge of the double blinding notebooks were photographed, the lab videotaped, and vials juggled and secretly coded. Randi even went so far as to wrap the labels in newspaper, seal them in an envelope, and then stick them on the ceiling so Benveniste and his team could not read them. The blinded experimental series showed no water memory effect. Maddoxs team published a report on the supervised experiments in the next issue July 1. Nature. 1. 4 Maddoxs team concluded that there is no substantial basis for the claim that anti Ig. E at high dilution by factors as great as 1. Maddoxs team initially speculated that someone in the lab was playing a trick on Benveniste,5 but later concluded, We believe the laboratory has fostered and then cherished a delusion about the interpretation of its data. Maddox also pointed out that two of Benvenistes researchers were being paid by the French homeopathic company Boiron. AftermatheditIn a response letter published in the same July issue of Nature, Benveniste lashed out at Maddox and complained about the ordeal he endured at the hands of the Nature team, comparing it to Salem witchhunts or Mc. Carthy like prosecutions. Both in the Nature response and during a later episode of Quirks and Quarks, Benveniste especially complained about Stewart, who he claimed acted as if they were all frauds and treated them with disdain, complaining about his typical know it all attitude. In his Nature letter, Benveniste also implied that Randi was attempting to hoodwink the experimental run by doing magic tricks, distracting the technician in charge of its supervision He was more apologetic on Quirks and Quarks, re phrasing his mention of Randi to imply that he had kept the team amused with his tricks and that his presence was generally welcomed. He also pointed out that although it was true two of his team members were being paid by a homeopathic company, the same company had paid Maddoxs teams hotel bill. Maddox was unapologetic, stating Im sorry we didnt find something more interesting. On the same Quirks and Quarks show he dismissed Benvenistes complaints, stating that because of the possibility that the results would be unduly promoted by the homeopathy community, an immediate re test was necessary. The failure of the tests demonstrated that the initial results were likely due to the experimenter effect. He also pointed out that the entire test procedure that Benveniste later complained about was one that had been agreed upon in advance by all parties. It was only after the test failed that Benveniste disputed its appropriateness. The debate continued in the letters section of Nature for several issues before being ended by the editorial board. It continued in the French press for some time,1. September Benveniste appeared on the British television discussion programme After Dark to debate the events live with Randi and others.