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Everything posted by BabcockHall

  1. Thank you; that is very helpful. I think that I had been assuming implicitly that the two antibodies (soluble versus immobilized) recognized the same epitope. I now wonder whether the antibody at the Test position versus the gold-particle labeled antibody recognize the same or different epitopes. Perhaps the answer is different, depending on which test is used.
  2. link Good Morning Everyone, I have been studying various forensic test kits for body fluids, and a number of them from Abacus Diagnostics or Independent Forensics use lateral diffusion immunochromatography. In a sandwich format the antigen forms a complex with both the soluble, labeled antibody and the antibody immobilized onto a membrane at the test (T) position. I can see how this could easily work with a protein such as hemoglobin, which is a tetramer of two alpha and two beta subunits. It is less clear how this assay works with human salivary alpha-amylase, which is a monomer from what I can gather. In other words I can see how the sandwich forms when there are multiple epitopes, but it is less clear to me what is happening when there might be only one epitope.
  3. Try expressing [ES] in terms of [E] and then proceeding with the differential equation.
  4. There is an alternate derivation that assumes that E, S, and ES are in quasi-equilibrium. It is actually an easier derivation than steady-state.
  5. You might look at the book Criminalistics by Richard Saferstein.
  6. SDS PAGE denatures proteins, and not every protein that is denatured can be renatured. If you need active enzymes and you want to separate by molecular weight, then gel filtration might be an appropriate technique. However, it is often the case that obtaining a pure protein requires more than one method of separation.
  7. My own experience is that the shape of the standard curve depends upon the identity of the protein. However I generally put little trust in absorbance values of greater than 1.0 in the Bradford assay, even using a research-grade instrument, let alone one that is not. The problem is that there is not as much excess Bradford reagent as one moves higher in protein concentration. A standard curve should be made, preferably with the same protein that is being assayed.
  8. As far as I am aware, protons only flow in one direction, outside to inside. The key to how the flagellar motor rotates in both directions may lie in the flexibility of FliG.
  9. I don't know much about MotA/MotB other than there was a nice genetic suppression study done on the generation of torque some years back. Can you explain what you mean by symmetric?
  10. My working hypothesis is that the flagellar rotor can rotate in either direction because FliG has multiple conformations owing to what might be termed hinges. I am unaware that ATP synthase has a similar property.
  11. Oguri K et al. (1995) Japanese J. Toxic. Environ. Health 41(4):274-279 were not able to isolate a cocaine-containing complex by silica gel chromatography. They suggested that the complex is too labile. However, they determined the stoichiometry between cocaine and cobalt to be 2:1 in one complex. They proposed a structure involving two bidentate cocaine ligands and two isothiocyanate (Figure 2 in their paper). My only reservation about their proposed structure is that the isothiocyanate may coordinate through nitrogen, not sulfur. I am still working on the acid-base aspects of this question.
  12. "An elucidation of the reaction mechanisms involved has been published [56] . lt assigns the formula (alkaloid H)2 [Co(SCN)4] to the relatively water-insoluble blue complexes formed in neutral-to-basic solutions, and the formula [Co(alkaloid)2] (SCN)2 to the more water-soluble, brownish-red to pink complexes formed in acid-to-neutral solutions. Solubilities in water and chloroform are given for complexes formed with 29 alkaloids and nine metal thiocyanates." Schlesinger Presumably (alkaloid H) is the protonated form of cocaine or other compounds. Experimentally, there are blue crystals initially formed in the reaction, then the solution turns pink upon addition of HCl and that addition of chloroform leads to a blue organic layer in the Scott test. What I don't understand is why the cationic species (the conjugate acid) is in the organic layer and the neutral species (conjugate base) is in the water layer. Yet all of the reference material (one textbook and a couple of articles) have stated some variation of the passage above. I am in the process of acquiring additional references, including (56) above. Does anyone have any thoughts?
  13. We are fairly early along in the overall project, and my thinking on this kind of testing is not overly sophisticated yet. However, it could be that qualitative results are acceptable at this point.
  14. Thank you for some food for thought. It seems to me that a volatile solvent (assuming it is essentially gone before the disk is put into place) solves many of the problems that you mentioned, possibly all of them. Or am I mistaken? We are in the process of making additional compounds and testing the ones that we already made.
  15. Inasmuch as we are working in collaboration, we are probably limited to disk diffusion for now. Because the compounds are soluble in DMSO, I was going to try acetone next for one of them. That way the acetone will simply evaporate, leaving the compound behind. The other compound came into a solution of K2HPO4. If we did our calculations correctly, we created a phosphate buffer.
  16. I am collaborating with a microbiologist to test some compounds to see whether or not they possess antibacterial properties. These compounds are either neutral or bear a single negative charge near pH 7, and they have aromatic rings. The compounds we have tested so far are soluble in DMSO, but modestly soluble in ethanol (less than 10 mg/mL), and probably not soluble in unbuffered water. The assay begins with adding the compound to a disk that is placed in contact with bacteria. My question concerns solvents that would be appropriate to use to dissolve the compounds. I assume that one control is to try the solvent without the compound of interest. Are there any solvents that are known to be so toxic in this sort of assay that they are best avoided altogether? If two solvents had about the same polarity, is it better to choice the more volatile or less volatile solvent? Is there any other property that we should consider? So far I looked in a microbiology laboratory manual, but I did not find anything. Perhaps there is a good reference that someone could suggest. Thank you.
  17. With respect to Q7, both RUBISCO and hexokinase can be eliminated from consideration because there is no net oxidation or reduction of carbon atoms and no photochemistry. They are clearly not part of photosynthesis. RUBISCO is obviously a key player in the Calvin-Benson cycle.
  18. "Reactions catalysed by enzymes follow saturation kinetics." The way I read this, it means that the Michaelis-Menten/Briggs-Haldane equation is followed. This is often true, but it is clearly false for many regulated enzymes (phosphofructokinase-1, for example), which follow sigmoidal kinetics. With respect to 4 if KM becomes smaller, then V/K becomes larger (keeping Vmax constant). V/K is the apparent second order rate constant for the rate when S << KM. It is also proportional to kcat/KM, one measure of catalytic efficiency. I agree with the answer in the key.
  19. What do you mean buy shorter reaction?
  20. You might give some thought to volatility.
  21. One of the reasons I am asking is because R2 is a little bit like electronegativity in chemistry; one teaches students about it, and they want to use it for everything, even when there are better tools. In this instance R2 is not ideal, because it is indifferent to the direction of the residual, only to its magnitude.
  22. I thank you both for some helpful comments. In our case we were plotting a straight line for gel electrophoresis data on proteins. The standard curve, which is mobility versus logarithm of molecular weight for the standards, had noticeable curvature. I am still looking into the biophysics, but the information that I have presently is that a slight deviation at high molecular weights is expected. I am not looking to explain the results, so much as to describe it, in the sense of making a more formal statement to the effect that a linear fit leads to non-random residuals.
  23. It has been suggested to me that Pearson's R is a good statistic for this situation. Thoughts?
  24. https://en.wikipedia.org/wiki/Anscombe's_quartet I would like to know whether or not there is a statistic that can differentiate between the case at the top left versus the top right. Clearly R2 does not do so. One could plot the residuals, and the non-random distribution sometimes becomes apparent. However, what I was hoping to find is some number, preferably one that would be calculated by a statistics program, that could be compared in the two situations. I am reading Motulsky's book Intuitive Biostatistics (that is where I first saw the Anscombe quartet, but I have not found anything in his book yet. I am presently using ProStat, which has both a calculation of COD (which I am pretty sure is R2), as well as a calculation of "Corrl" which is said by the user manual to indicate "how closely the two variables approximate a linear relationship to each other." I note the presence of squared differences in the numerator of COD, which are not found in Corrl.
  25. The APS was fresh, but the first bis that I used was old. There is also a chance that I made a gross error in weighing it out.
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