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Xray-based Protein Visualization

Easier than current methods.
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Proteins are the little molecules that (for the most part) "do" everything inside of cells. Combinations of protein interactions are the de facto mechanisms by which individual cellular "behaviour" is conducted. Large groups of cells working together, or in a similar fashion, are the mechanisms of tissue function...tissues combine for organs or system function, and so on...all adding up to give you the ability to read this idea :() . All starting, mechanistically speaking, from proteins in individual cells whizzing around and doing stuff. Not surprisingly, since proteins are the molecules that do stuff, in trying to understand how we work, researchers often look at what proteins are used and when, when studying virtually any biological phenomenom (All diseases, neuroscience, development etc). There are many methods to do this, but primarily people use two:

1. antibody based evaluation methods, in which an antibody you wash over thin sections of your tissue of interest, hunts down and attaches to your protein and gives off a signal (usually light). If you see light, you that you know your protein is there in that tissue at that time, which may give you a clue about what your protein does, or how the cells, or tissue works. This technology has been around in various forms for almost thirty years.

2. Genetics- Proteins can be altered before they are actually made, by messing around with the DNA of an animal or cell. By taking out or adding certain portions of DNA, you can alter the function of specific proteins you want to mess with, which may or may not mess up the cells, other proteins, cell division, tissue function etc. All of these give you a clue about what the protein does, which hopefully is important in some way.

In mammals, except in embryos, evaluating individual protein expression patterns is done by tissue sectioning and for the most part described qualitatively. This can be time consuming for the process, inaccurate, wholly dependent on the anti- body properties, and often generates so much visual data that much information is lost.

If there was a rapid method to evaluate tissue protein expression patterns in 3D, in situ and quantitatively, it would speed things up. The idea below is sort of like an Xray version of green flourescent protein. If we could visualize protein expression in situ with Xrays we could have a means by which to evaluate the 3 dimensional protein expression patterns.

Current research Xray technology (called Micro ct), widely used to study bone allows visualization down to 10 microns per voxel, this is roughly the size of two cell nuclei. The problem is that most tissue is too soft and the xrays shoot right through it, so no signal. Their are some stains methods to visualize soft tissue differences, osmium, iodine , phosphotungstic acid, which differently bind to tissue types, but these only enable visualization of tendons, bone, mesenchyme, follicles and epidermis.

In order to look at protein expression patterns with Xrays, I propose to make transgenic mice that express Iodine Binding Factor, a monomeric water soluble protein from mature grain which preferentially binds iodine, under "whatever protein's" promoter. Upon animal sacrifice, treatment with Iodine, which easily diffuses through tissue, will bind to the tissues that express Iodine Binding Factor, indicating your 3D spatiotemporal protein expression pattern of your gene/protein of interest. These Iodine laden tissues should then be dense enough to give a nice quantitative 3D expression pattern, that can be visualized with Micro ct. All without antibody-based methods or sectioning.

Ups: Easy visualization, Quantitation, Iodine Binding Factor is a grain gene not expressed in mammals so no putative physiological role, saves time.

Downs: 1 protein at a time, due to resolution issues, it could only evaluate at the tissue level (couple hundred microns, good for developmental and differentiation research). Iodine Binding Factor expression could disturb tissue function, but there are CRE systems these days to activate gene expression postnatally, so this could be avoided.

leinypoo13, May 12 2009

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       This is not as dumb an idea as I thought it was going to be. But: (1) can you get a useful absorbance with the amounts of protein (and hence iodine) in question? Most proteins are present at very very low levels. (2) you'd really want the iodine-binding moiety in the protein you were interested in (a bit like including a GFP moiety in a protein), rather than just expressed under the same promotor; different genomic locations give different expression. (3) why not go for something that chelates some heavy metal ion? I'm guessing it would be more absorbative than iodine (but I'm guessing. Oh, I just said that.)
MaxwellBuchanan, May 12 2009
  

       Just back up slow and put the bone on the floor.   

       To your queries, I say: don't know, o.k., and not sure. Maybe worth the effort to play around with though, a big payoff if it works.
leinypoo13, May 12 2009
  
      
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