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Max_Normal

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  1. Whether you would easily determine this using Western Blotting would very much depend on your protein. You need to provide more information. What is your protein of interest, what is the splice variant, what is the species, do you have accession numbers (preferably NCBI ones), were you using denaturing SDS-PAGE gels, do you have any information on the functional difference between the splice varients. Briefly from what you have given me though, have you checked that the different between the two mRNAs is in the ORF and not in one of the UTRs? If a UTR is all that is being spliced, your protein size will be the same in both variant but your mRNA will be different. Also, unless there is quite a bit of difference between the variants (at least a Kda or two), you might find that it is hard to separate them using SDS-PAGE unless you use very high or very low % gels. If the kDa is middling, you'll find them even harder to separate. Protein folding should not be an issue if you are using denaturing gels. Have you checked the epitope that your Western Blotting antibody was raised against? If you are using a monoclonal, or a polyclonal raised against a peptide instead of whole molecule, you might find that your antibody epitope does not exist in your splice variant and that's why you don't see it. If this does not help, come back with more info and I'll have a better look at it
  2. 2 easy ways to test this, obviously an antibody to ubiquitin, but if you treat with the proteasome inhibitor mg132, you might see a build up in intensity of your heavier band along with the disappearance of the third band if it is a degredation product.
  3. Are you using this for nucleic acid separation? I use agarose myself, but I thought that 6% to 12% was a normal range for a nucleic acid acrylamide gel. I use acrylamide for protein, just 4% to 17.5% depending on what i want to see. 40% just seems very high percentage, I have never heard of it being used straight out of the bottle, have you checked your protocol?
  4. Looks like a massive shift just to be down the the charge on the glutathionine doesn't it? From what I remember, this just gains or loses a single proton, so the shift would be relatively small. If the protein was undergoing multiple glutathiolation (or phosphorylation), you'd perhaps to see more of a ladder of differently migrating bands. Perhaps that's what the third band shows, the difference between zero, single or double glutathiolation? Are you certain that this is not a splice variant or isoform that is being differentially expressed as a result of your treatment? Have you considered sumolation and ubiquitination? Your band shift is not far off the size of ubiquitin, and this is a covalent modification that is resistant to reducing conditions.
  5. The standard transmembrane ER marker is Calnexin. It's a medium sized protein, and should be easy to clone if you are making a GFP chimera. Don't confuse this with Calregulin, which shares a lot of homology but is lumenal rather than transmembrane. The sequence will already have an SRP insertion sequence, I expect, so you should be OK with the GFP on the C-terminus. If you just need to visualise the ER (although this will not be membrane associated, so presumably you don't, unless you just hate yourself), a good way is to simply add a KDEL sequence to the N-terminus of the fluorescent protein. KDEL-GFP will be continuously recycled through the ER and you will get a nice reticular stain. In fact I have one of these in RFP and one in GFP, contact me if you do want an aliquot, and i'll post it. Alternatively you can use lipid based markers such as C5- and C6-ceramide-BODIPY, these will insert into the ER/golgi membrane (make sure you choose which one is enriched in the ER carefully). Other than that, you could clone of the proteins that makes up the signal Recognition Particle that the nascent peptide is extruded into.
  6. P.S. for your flukes, check out beef tapeworm, I think that is what you are looking for........ Fresh raw beef is pretty safe, although not such a good idea for burgers.
  7. Blood (and therefore muscle tissue like steak) never, ever, ever contains bacteria unless you are sick! Any bacteria that get in though a wound get mopped up locally and pretty quickly by your immune system. Any bacteria in your meat or burger came from after the animal died. Your digestive system is absolutely jammed with bacteria all of the time, many of which you need to digest your food properly. Most pathogenic bacteria you eat are killed by the HCl in your stomach, but there are some that can resist very low pH (such as H. pylori that causes heartburn). There is a lot of confusion of which is "good" or "bad" bacteria. For example everyone has E.coli in their gut, but you wouldn't want it in a wound as it can cause gangrene. Most bacteria are opportunists, and you can live with them for years until they get in the wrong place or gain a growth advantage over other species of bacteria, and then you get sick.
  8. Actually this is a gel rather than a bio-paper. Printing cells onto paper will cause many problems as they have to be in an aqueus medium (to allow diffusion of molecules to the cell) that contains:- A) A pH buffer to counteract the lactic acid buildup from cellular respiration. B) Amino acids to facilitate the constant protein synthesis that cells need to survive and C) Growth factors (hormone-like signalling molecules) that constantly tell cells to divide, (MAPK signalling) investigate the other cells in their environment and keeps protein synthesis from shutting down (mTOR signalling). Usually supplied from blood serum from calf foetuses. D) Glucose to facilitate respiration. Therein lies the problem, with no immediate vascularisation (blood vessels with flowing blood) to provide the above mentioned conditions, cells with rapidly die. This is why if you let a cell culture grow more than 2 or 3 cells thick, they start to die off - they simply don't get enough access to the nutrients. This is why we do not have synthetically grown organs (or steaks, yum! fetal calf serum flavoured) already. It's a fantastic idea, but far more advanced than current cell culture technology. If you could incorporate a blood capilliary network into the tissue as it was printed, and rapidly hook it up to a blood or tissue culture medium supply, you'd be on to a winner. Until then, no go. Bear in mind also that tissue culture has no immune system, so all of this has to be done under absolutely sterile conditions. Very hard indeed.
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