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Everything posted by iRNAblogger

  1. So I don't have any direct experience with this topic, but from my reading, I would start with a trypsin digest of the protein. You can look at this paper to see the basics: http://www.ncbi.nlm.nih.gov/pubmed/3930949 Another method is using cyanogen bromide. If your protein has any methionines in it, then you can get relatively large protein fragments using this chemical for cleavage. The two ways to characterize the peptide fragments would be Edman Degradation or Mass Spec. Or if all you are looking for is simple information such as "how many methionines are in this protein" then you can use cyanogen bromide and simple SDS-PAGE to figure that out. If you are curious how many lysines or arginines, a similar experiment using trypsin and SDS-PAGE would work. But it depends on what kind of information you are looking for.
  2. Are you concerned with the typology of the plasmid? (Such as amount of supercoiled vs relaxed vs nicked?) Because some studies have suggested that temperature changes can affect the supercoiled state of plasmids: http://www.pnas.org/content/81/13/4046.full.pdf+html and I'm sure that a similar phenomenon could be observed due to freeze-thaw cycles.
  3. Yeah, a suppressor mutant screen would be a pretty good way to start depending on your model organism! I also agree with chadn737 that a pull-down of your kinase is something else you can start with. But if you have a good idea of what type of protein you are looking for, you could try to use bioinformatics to predict a set of proteins that are putatively involved, and then follow up with knock-outs or knock-downs to see if they are actually involved in your pathway. You might also consider a yeast-2-hybrid screen for interaction partners. It may prove useful to take a look at this article: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0065011 Additionally it sounds like you have some predicted targets for the initial kinase, so if you know anything about how these proteins are specifically modified by the kinase, you can try to analyze phospho-negative mutants (mutate the phosphorylation target serine/threonine to alanine for s/t kinase or tyrosine to phenylalanine if the kinase is a tyrosine kinase) or phospho-mimic mutants (by mutating serine/threonine to aspartate or glutamate, if your kinase is an s/t kinase). Hope that helps! It would be nice to know what your model organism is, too. That might help determine what your plan of attack should be.
  4. Sorry! I guess I misspoke. The electrons are not freed and then absorbed: is rather that the chlorophyll is oxidizing the water (taking the electrons directly from the water). Is this more correct? Also I noticed thomaszp that you were interested in the the way the electron is carried and transported. You mentioned resonance, which is important for the photon absorption (this is because the organization of the double bonds creates a conjugated system which is easily excited by light). However, would it be accurate to say that it is actually the Mg atom which carries and transfers the electron (going by its changes in oxidation states, mentioned by CharonY)?
  5. Oooooh, a list like that would be really nice... I haven't come across anything like that before (there are so many different commercial reasons and ways that you could immobilize an enzyme, that I doubt anyone has compiled a list like that, maybe there IS one out there though!) I would do more of a guess-and-check approach. Try thinking of enzymes that might be useful in an immobilized state and then do a search for any instances in which it has been immobilized. I will keep my eye out for a list though!
  6. That's a good point made by ewmon. I think that is the idea that Miller and Urey were beginning to get at with their "primordial soup" experiments. However, I'm not sure I would be comfortable calling the ocean one giant life form; this gets back to the age-old question of how do we define life. But you could definitely make the argument that one possible hypothesis is that biomolecule precursors were present in the ocean and then became encapsulated in protobiotic forms, which eventually evolved into the life that we observe and describe today. But to comment on the article (very interesting by the way!), I didn't see an academic article citation, so I couldn't look at the methods in the paper to answer this question. Does anyone know if the scientists accounted for intermolecular forces in their probability calculation. I have a feeling a probability calculation is not the best prediction for which molecules could be trapped in the liposomes: there are a lot of complicated interactions among different biomolecules that cause them to associate and aggregate with each other, something that would be extremely difficult to predict statistically.
  7. Well, I don't think you could design a specific primer because of the genetic code redundancy. BUT you could design a collection of primers based on the amino acid sequence allowing for each of the possible amino acid codes. However, you would have a TON of primers, and you might also lose hybridization specificity.
  8. When you say allele specific primers, are you referring to allele specific oligonucleotides (because if not, then I would suggest this method! There's a slight semantic difference between primer and oligonucleotide, which has to do with how you use them). Other than that, you're likely going to have to do a lot of sequencing (you could amplify a really short fragment to speed up the process, as long as you know exactly where the SNP is supposed to be).
  9. That's a good question. I have never heard of an enzyme that specifically targets the sidechains of a protein's amino acids. However, to begin the search would look at different metabolic enzymes (which might be better prepared to destabilize and cleave C-C bonds) or deaminases (which might be able to take off that ammonia from lysine or the guanidium from arginine). I'm not really sure though. I am also skeptical that these enzymes would be able to work on a protein, I think it would be more likely that they acted on free amino acids or very small peptides (I'm mostly thinking of steric interactions that would prevent an enzyme from getting at the individual side chains on a protein....).
  10. So I think the source of your confusion is that you missed a critical step of the light reactions: the splitting of water. The initial source of "free" electrons in the system is the water molecule which is split by photosystem II. Once the electrons are "freed," I THINK that they are essentially "absorbed" by chlorophyll, which when excited by a photon, is able to channel them through the electron transport chain in order to produce ATP. This explanation is how I think about the light reactions, but I'm definitely not an expert.
  11. If you are looking for reliable information, I would suggest you go directly to the scientific literature (it's hard to tell who has authority on the matter on a forum like this, and it's probably not the best place to get medical advice). In what context are you looking for "effectiveness"? There are studies covering the effectiveness of magnesium supplementation on ADHD, blood pressure, pregnancy, etc. Let me know so that I can point you in the direction of some good sources (so then you could draw the conclusions yourself rather than depending on unreliable sources)
  12. So I don't think that you are supposed to get answers for test questions on this website (so I'm not sure if I'm "allowed" to answer this, so I will try to only nudge you in the right direction rather than give you a straightforward answer). What would happen after the double-strand break? What's the next step? (I can tell you that it would be none of the mechanisms you mentioned) Think about the different DNA-repair pathways to answer the question of how double-strand breaks are resolved. What other approaches could you use that don't necessarily involve any of the mechanisms you mentioned? What other processes in the cell could lead to sequence repeat instability? I have a possible answer in my head, but I'm not absolutely sure. Sorry for being so cryptic in my response!
  13. So, in my opinion, bugs are awesome! I love to watch them, but I don't really enjoy touching most of them. I try not to kill bugs for the most part mostly because even though we don't immediately see the effect, our selection against certain types of bugs definitely introduces a selective factor into the ecosystem (even if it seems negligible!). Also, I strongly believe that bugs have the ability to feel: they respond to touch, ever tried to pick up a worm? If they can feel pressure, why wouldn't they be able to feel pain? They definitely have nervous systems. Anyway, I try to leave them alone, so long as they aren't endangering anything.
  14. I agree with CharonY. I would like to note, however, that the purpose of the bacterial capsule may be very diverse for different species, and it might be difficult to give a single answer to the question of its function. The purpose of the Gram stain is to characterize bacteria based on its cell wall composition, but deeper than characterization/identification (ie. function), you would have to study more into the behavior of the bacteria. Also, to answer your second question about how the Gram stain works. As CharonY mentioned, the crystal violet is unable to be adsorbed by the gram- bacteria. This observation is likely because crystal violet contains three N atoms, including a charged group, which would result in high affinity for the sugar structures in both the peptidoglycan and lipopolysaccharide layers. However, when the decolorizer step is applied, the lipopolysaccharides appear to fall off of the cell wall, thus causing the bacteria to lose the capsule. The counterstain works much the same way as the crystal violet, but if the cells were already stained blue/violet on the peptidoglycan level, then the stain won't show up as much. Does that make sense?
  15. So I have a couple guesses, but that is pretty weird. 1. MSA does inhibit the growth of gram - bacteria, but that doesn't mean that every single gram - bacteria in the world would be unable to grow in those conditions, there are probably some exceptions. You may have found an exception. 2. The bacteria might have an EC matrix that is heavy in lipids while also containing a thick peptidoglycan wall, which would allow it to survive on MSA, but then also prevent it from staining purple. 3. A simple contamination issue. You might have accidentally contaminated your TSA plate. If you were to re-do the experiment, I would streak onto an MSA plate for the growth step rather than TSA which is not selective.
  16. I am experiencing CharonY's confusion as well. What exactly is the purpose of your initial 3-hr incubation step? Generally incubation is supposed to refer to growth conditions, but if you are incubating the bacteria with a ligand or something, then it MIGHT make more sense. I would suggest that for a CFU calculation, all you should be doing at the bench is diluting out your bacterial sample, spreading on plates, incubating (at optimum growth temperature), and then counting the colonies. Are you testing the CFU after incubation with a ligand or substrate or chemical of some sort? That might be the source of the problem.
  17. Another possibility as your solvent is phosphate-buffered saline, which is easily accessibly and widely used when working with tissues in vitro. I don't THINK it would cause any interference, but my physics knowledge is limited...
  18. I just have a quick question. What type of enzymes were the urzyme descendants? It says here that the enzymes "translated" the genetic code. This claim, semantically, refers to the process of translation, which is not catalyzed by protein enzymes. It could, I guess, be referring to transcription, which in the public sphere might be better understood as the act of "translating," which would make more sense because it is catalyzed by a protein enzyme. Also, just to play devil's advocate a little, I didn't think that the RNA world hypothesis claimed that life was exclusively RNA-based... (maybe I was just making that up). I always assumed that the RNA world hypothesis did not exclude proteins, but rather that it simply suggested that A major catalytic molecule (not the only one) at the time might have been RNA. But I guess I had always assumed that the fact that RNA catalyzes protein synthesis indicated the possibility that both were hypothetically part of the ancient world.
  19. So long as the tea leaves do not look like that when you first ingest them, I think you should be fine. We accidentally ingest all sorts of fungi and fungal spores throughout a normal day, so unless there is a particularly high number of fungal cells on the tea when you drink it, it should be safe. If you are worried about it, you could take the tea leaves, spread them on a cookie sheet and bake them in the oven briefly just to kill everything (this may cause the tea to taste weird, I've never tried it before)
  20. I agree with CharonY (BTW CharonY, you are awesome and have an answer for everything!). I would like to add that more generally, these organelles were first observed through a variety of ways. They were isolated by cell fractionation, they were observed under the microscope, and they were characterized based on their morphologies at first. Nobody had any clue what these organelles really were (or what their function was) until more in-depth studies were done. This idea is something that I personally really struggled with in my biology education. I had always been slightly confused how textbooks obtained the authority that they seemed to have to make claims, such as "the mitochondria are the powerhouses of the cell." It wasn't until I started finding that there are whole books recounting the discovery of certain phenomena that are subsequently condensed into one sentence in a biology textbook that I began to understand that a textbook is a very brief summary of the discoveries made on these topics. While it is very helpful to see big-picture stuff, it is also important to return to the primary scientific literature to see where these assumptions come from.
  21. Yes. I agree with Ringer. A test cross is between an unknown dominant genotype and a homozygous recessive in order to determine if the subject is heterozygous or homozygous dominant. I have never worked with mice, so if I were you, I would look at the literature. See if, in the methods section of a paper, they mention "out of ___ number of progeny, _____ number were this phenotype and blank number were this phenotype, so the individual was deemed ___ genotype." You may have to dig deep into older literature, because I don't think this is very common anymore to include in papers.
  22. Also (to add to the concerns above), "editing" a genome of a person or animal that is already past the zygotic stage is virtually impossible. We wouldn't even get to the stage where we could worry about negative effects of having the gene integrated into our genomes. Occasionally this phenomenon does occur, and it more often than not results in cancer. In order to "edit" a person's genome, you would have to transform that person's cells with 100% efficiency (at this point in time, this is absolutely impossible). In addition to transforming the person's cells with 100% efficiency, you would need to expose each and every one of that person's cells to the transformation vector (virtually impossible). Even if you could accomplish these above steps, it is extremely unlikely that you could ensure integration of the gene in a stable location (it is likely that you would integrate into another gene, which could result in lethality or cancerous growth for that particular cell's progeny, or into a silenced area of the genome, which would result in non-expression). You WOULD be able to do this "editing" if you had access to the zygote or gametes (pre-fertilization) and could perform the necessary transformation procedure and then screening process (which is how it is done in mice/zebra fish/other lab animals and plants). However, it is generally considered unethical to genetically transform a human zygote due to the concerns brought up by previous answers. Perhaps in the future, when science technology has far, far surpassed that of today, will we be able to consider genome "editing" of humans.
  23. The genetic approach to your question would be to employ plant transformation methods (as mentioned by CharonY) such as Agrobacterium-mediated transformation or, alternatively, a so-called "gene gun." The short answer to your question is that getting the plant to express this RFP in its petals is absolutely possible, and I don't think it would be a difficult thing to do given the resources, time, money, and experience. However, transfection is a complicated business and requires a lot of trial-and-error (and a TON of post-transformation screening because these gene insertions are random!). But, yeah, it's totally do-able in a lab. However, it sounds like you are talking about a home-setting (and I strongly advise you against attempting to transform your plant in your home). If it is true that you are in a home-setting, I would definitely recommend the dye idea.
  24. I have mixed feelings about Monsanto's approach to agriculture. While I am not really against the genetic modification of plants (I think it has some pretty important uses in today's society, and it appears to be harmless to humans) I don't see it as a sustainable approach to agriculture (the fact that most [or all? not sure] GM plants are sterile prevents farmers from diversifying their crop based on artificial selection of desirable traits. Instead of choosing which corn to collect seed from, the corn that is disease- or cold- or drought-resistant, the farmer now chooses from Monsanto's selection of disease- or cold- or drought-resistant seed. This system is much more efficient, I suppose, but it doesn't appear sustainable). We will have to see how the practice of agriculture continues to evolve with access to GM crops. I think that we have seen the beneficial side of GM crops, increased yield, decreased losses, etc., but now we will soon begin to see the consequences, if there actually are any. Also I totally agree pippo, it does sound like Star Trek!
  25. Hey, there is a simple solution: incomplete dominance! This phenomenon is when the heterozygote (in your novel, I guess this would mean the EF individuals) exhibits a phenotype somewhere intermediate to the dominant and recessive phenotypes.
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