CharonY

You generally use polyclonal antibodies as second ones. These happen to be IgGs

That's rather unlikely. Just by browsing I do not see an awful amount of paper on whales and many of them are using molecular biological techniques. In other words, you only need a couple of cells to do the respective experiments, slaughtering a whole whale is, in the truest sense of the word, "overkill". Also apparently a lot of the meat that is not getting eaten simply lie rotting around: http://www.news.com.au/adelaidenow/story/0,22606,23206289-5006301,00.html Finally you may want to read the following article: Parsons et al 2006, Marine Pollution Bulletin It’s not just poor science – Japan’s “scientific” whaling may be a human health risk too

Yes, that's why it is important to include statistics

So do you mean that you made a run with various concentrations on a single day and then repeated the whole assay on another (i.e. there is not likely a bias towards specific concentrations?). Regarding the analysis, "the error bars are pretty tight" is at best just a qualitative assessment. At the very least do a t-test.

Before you proceed thinking in that direction have you double checked that your results are statistically relevant? Moreover are the results repeatable? Was everything standardized? That is, where the experiments done in one go, or on different days? Where the cells from a single split culture or could they have different histories? Whenever something counterintuitive pops up it makes sense to be really really sure that the effects were not due to handling, different batches of cells etc. Otherwise you are going to lose a bunch of time.

Well, basically it is not uncommon to have concentration dependent effects. It is kind of strange that a high amount has seemingly beneficial effects, though. In general, concentration-dependent effects can be unspecific. E.g. w/o knowing details one can speculate that the redox enzymes decompose and increase the amount of copper in the medium. However, I'd not assume that this would be beneficial (an iron enzyme might make more sense, depending on the cell culture). One question would be how this bacterial proteins is supposed to affect the growth of the cell line in this particular experiment?

Unfortunately there are a gazillion possibilities why it does not work. First question is whether there is production of the correct protein at all. Based on the OP I'd assume that there was low production. Have you checked whether it is the correct protein (e.g. by MS/MS)?

As I have mentioned above, rRNA is often isolated by microbiologists interested in the active microorganisms in the environment. RNA degrades much faster than DNA, so if you only go for the DNA chances are that you will also have many sequences of bacteria that are long dead. Everyone amplifying rRNA sequences will at some points notice that: if you do not work in a clean area you might get amplifications using universal rRNA gene primers even if no template is added (one of the reasons why these bothersome PCR cabinets are used). If you isolate RNA on the other hand, chances are higher that you will get sill living organisms. In other words, you will get somewhat different results if you amplify directly from DNA as opposed to RT-rRNA. This is of course only the case in complex communities. If you got some highly enriched samples or even pure cultures it does not make sense to go for the RNA.

It could very well be a respect thing. I noticed that the staff here (in Arizona) always refer to the professors as Dr. something, whereas postdocs are addressed without a title, for instance.

Actually you are right to consider how the antibodies were produced in the first place. It is e.g. a difference if the protein or only the relevant peptides were presented (or if the design was first done in silico). I assume that in this case the antibodies in question have never before been used in a Western? Of course one can try doing a non-denaturing gel, though the resolution would be rather bad. Anyway, APCs do not denature proteins (as antigens) in a similar way as SDS does, but they degrade it. So in the end they would present peptides. A discontinuous epitope of that antigen has to remain, per definitionem, intact. Otherwise it would not be an epitope, nor would it be considered discontinuous.

To vbreemars, just to clarify: 27F and 1492R are universal primers, these are conserved regions of the 16s rRNA gene. If they were specific primers you would only get amplificates from a single genus (or even species). Anyway, as I mentioned above, both approaches are viable. RNA has a faster turnover, though, so microbes detected that way are more likely to be still active. What some do is to do both, used DNA and cDNA to analyse the differences between those two pools.

I am not sure what your question is. Proteins of the Lrp-family bind DNA via a N-terminal HTH motif (pretty much standard in that regard). Generally, if you find that a particular paper is missing information that you need, try to track down a review. There are a couple around of LRP-type proteins. If you are interested what the Lrp-protein in E. coli does, there are number around describing its regulon (it is rather a global regulator IIRC). Or are you actually interested in the particular mode of the Lrp-regulated Pap-pili switch? That is, how methylation is inhibited?

Essentially that is all what systems biology is trying to achieve. However it won't be faster than traditional wetlab work because modeling is dependent on good data: garbage in garbage out. At the moment most believe that it will take at least 50 years to get the complete metabolism of a single cell seriously modeled.

Quoted for importance. Neglecting mathematics might give you problems later on. At the moment you can in theory get a phd in bio (in fact, with the current system once you are accepted as graduate student you will almost always get a phd, no matter what, but that's a different issue) without proper mathematical training, but it will be to your disadvantage.

Actually to me this part here: implied that the it was assumed that continuous epitopes are detectable but the question was what happens if it's not.

Actually SDS treatment will have effects on the efficiency of subsequent antibody assays. In case of linear epitopes the binding efficiency might be enhanced. Discontinuous epitopes, however are a tricky matter. In most cases denaturing electrophoresis is not recommended in such cases. Sometimes it does work, possibly due to renaturation. I also believe that strong disulfide bonds can stabilize the epitopes, depending how close the involved aas are within the protein. So in short, Western blots are not really suited for most discontinuous epitopes. I actually recall there being a paper a year or so back in which surprisingly discontinuous epitopes were detected in Westerns. Maybe I can dig it up. Just to clarify a point though, the epitope to be detected is clearly a discontinuous one? Or has the AB just been initially raised against one?

Ah, it is quite common that one gets confused about the directions of the DNA coding strand. However, a simple way to memorize it is that the polymerases move from 5'->3'. The reverse transcription is no exception, it is simply a RNA dependent DNA polymerase. Transcription of rRNA genes works precisely the same way as for mRNa, only because it is a structural RNA does not change the transcription method. Regarding the sequencing reaction: first note that due to technical reasons you usually do not sequence a single strand. Normally after the RT you amplify the ssDNA by PCR and thus yielding dsDNA again. However, the direction of sequencing (whether you are on the codogenic strand, or not) is dependent on the primer that you use. So if you omit a PCR step and directly sequence the single strands directly from the RT, you'd get the codogenic strand. I gather from those questions that you ask why people actually bother to sequence the rRNA as opposed just to isolate DNA? Let's start with the latter questions. Bacteria do not splice and do not have introns. So this is obviously out of the way. Now there are different reasons why you would be more interested in DNA instead of RNA. The most obvious one is that ribosomal DNA is more stable than the rRNA counterpart. As such by DNA profiling you will also find RNA genes of inactive organisms. Moreover, you can tag and separate rRNA of metabolically active organisms (e.g. via stable isotope probing), which you cannot with DNA.

The easy bit first: the A250/A230 ratio suggests guanidine isothicyanate contamination (from the Trizol). To clean that up add another precipitation/ethanol washing step. Regarding the additional band: it is hard to judge w/o really seeing the gel. However, note that of course chromosomal DNA, especially if sheared, will not have the correct size as indicated by the marker. The marker is only accurate for completely linearized DNA. Also, did you use a denaturing gel and how long was the DNAse treatment? For better troubleshooting the complete protocol as well as a picture would be beneficial.

Yep, that is one of the differences I noticed here. In Germany a professor is comparable to a department head. E.g. where I did my phd my prof had ~150 people (phds, graduate students, undergrads and staff) working for him. He was the only one possessing the title of a professor out of the whole bunch. As such a German professor is way high in the academic food chain. In the US and I suppose also in the UK a professor is more a teacher. That's kind of the things that appear to be similar internationally but in fact are not. Got me kinda irritated that everyone here was called professor until I noticed that they weren't even tenured ;P

Almost all PhDs I know (including myself) do not use the Dr. It is sometimes used when one looks kinda youngish and gets introduced to other senior scientist with whom one is not really familiar with. This is just to distinguish PhD students from the postdocs but after that one tends to be on first name terms. I actually ever only use my Dr. while communicating with medical doctors. Otherwise I never use it, even not with students (well, unless someone particularly gets on my nerves). On the other hand, senior professors are usually addressed as Prof. or Dr. by students anyway, so they usually do not need to enforce it. In Germany there are no assistant or associated profs, btw. So only the department head gets the title of a professor. From what I have seen in the US so far professors are called Dr. by the staff and students up until they start working in their labs. Then it is often first names again (or at least last name w/o Dr.).

Basically you can assume that every portion of your body that is sufficiently wet (yes, including the stomach) harbors bacteria. If they are not pathogens it is generally a good thing as they limit the resources for pathogens by merely being (and living) there.

Some good points were put forth here. Basically the textbook definitions of life (usually at least requiring a membrane enclosed plasma, metabolism and the ability to reproduce) are more a description of known life forms than a true definition applicable for the actual identification of life. This is a severe limitation as definitions go. In the end it borders more to a philosophical question as ultimately "alive" is not a measurable quality but rather a denominator of a bunch of qualities that we intuitively associate with something we call a living organism. In practical terms, though, there are not many examples of "borderline" examples that exhibit only some of the ascribed properties. The most known ones are viruses that don't have metabolism nor membrane (capsules usually do not count). And just as a sidepoint, someone mentioned that respiration falls under excretion (I think). It does not. Now carry on

A number of intracellular proteins are glycosylated and the function of the glycosylation are manifold. They can , for instance, be necessary for intracellular trafficking (in eukaryotes, of course). Also, many signal molecules are dependent on correct glycosylation. One can, of course argue whether it is intra- or intracellular signalling (dependent on what step you look at). A classification is also complicated for glycosylated DNA binding proteins. Technically they are not necessarily signaling molecules, however the glycosylation might affect their binding ability and as such there might be changes in gene expression levels, for instance.

OK, melting point analyses won't give you much information as the 16s region is highly conservered and you will not see noticeable shifts between most bacteria. What you want to measure is the fluorescence curve obtained in from your machine. If the product is amplified you will see an increase in fluorescence. You will then determine the crossing point to determine relative abundance. In order to see what bacteria you got you'll have to sequence the PCR products and build trees from the sequence alignments. Alternatively you can use highly specific primers (but then you might run into trouble if you got bacteria with yet unsequenced rRNA sequences.

Do you want a list? Also, for quite a number of modifications, involvements in intracellular signaling have been proposed, but not conclusively shown.