CharonY

With 20 kDa G-25 is a good bet (cut-off is around 3-5k, IIRC). G-50 is about 30k, which would not be useful to you application (assuming globular proteins that is). Maximum volume is directly dependent on the pore volume (and other factors such as particle size as it affects sample dispersion and thus peak width).

Is this a homework question? There are quite a number of suitable experiments. The general elements are the modification of the elements of interest (most cases that would be mutagenesis) and monitoring the outcome (for regulatory elements it could be expression studies, for example). For protein-protein interactions there are also many techniques available, depending on what you want to see at the end and what kind of instrumentation you may have available. Considering that this may be related to some kind of course you should probably just look at the techniques being discussed to find the suitable ones/

Well, both are linked to cancer and are known to be mutagenic. Due to the way the respective compounds are taken up and metabolized, some tissues/organs tend to be more affected than others. That said, tobacco consumption has been found to be implicated in buccal cell changes (for review: Proia et al. Cancer Epidemiol Biomarkers Prev. 2006 15(6):1061-77). I am less certain about alcohol, especially as it is converted to acetaldeyde before being genotoxic. From that I would assume that during non-excessive alcohol consumption the effect on buccal cells could be rather low..

Oral tolerance is, to my knowledge, only partly understood. The common part is that both antigens (food and pathogens) are presented to T-lymphocytes, however the way they are presented changes the antigen-specific responses.It has been found that in absence of inflammatory signals, presentation of antigens to T-cells by dendritic cells favors induction of tolerance rather than activation. The precise molecular reasons are (again, to my limited knowledge as it is not my field) not quite clear but IIRC numerous activities in the spleen, and various lymph nodes as well as the lymphoid mucosa were found to be involved. Another route is the presentation of food antigens by enterocytes which, in absence of further stimulating molecules causes the T-cells to be unresponsive to further exposure to the antigen (anergy). I am pretty sure that more up-to-date research should be available, if you look for articles about oral tolerance.

Excellent point. I (obviously) have not thought of that.

You mean there is a fungal infection, or how did you determine that a) there was yeast and b) that the adverse effect was by nutrient competition? The reasoning is likely to be off, indoles do not "produce" yeast. There are plenty of sources very fungi can come from, especially if you do not work under sterile condition. However, if you did your own nutrient solution under non-sterile condition, there is a good chance that you accidentally cultivated fungi in it and then infected the plant with it.

I never mentioned metabolic independence. In fact, interdependence is quite common. The distinguishing factor is that viruses are unable to do any type of metabolic activities on their own.

I do not think that one can reasonably assess that. Many heart attacks are not associated with pain, for example http://www.nhlbi.nih.gov/health/health-topics/topics/heartattack/signs.html

Which metrics did you use to assume that and what are the comparative scales? I.e. do you compare biodiversity say, per area unit or per country (that is, political borders)?

Injecting foreign antibodies will elicit an immune response (that is how secondary antibodies are raised). AFAIK there are trials of using transgenic mice to produce human sequences. I think some of them even went to the trial phase, but I am not up to date on this matter. However, these are supposed to be used for therapeutic purposes and I would be surprised if that would be able to be diverse enough to replace a complete immune response.

Rare relative to what?

It depends quite a bit on the protocol (and kit components). I always made the stains myself and use the MS-compatible staind. One of the first things to go tends to be the sensitizer. If made yourself or the bottle has been opened (but stored appropriatelyI would think that within a few months the components would degrade significantly. Fully closed kits tend to have shelf lives of 1-2 years (though I would check the manual and lot information). 3-4 years may be pushing it (especially if they have been around a while before purchase), but I would simply run a test gel with a dilution series of a reference sample and look how sensitive it still is and whether it is sufficient for applications.

It is also quite a bit dependent on perspective. Scientists from different (sub-)discipline. From a physiological perspective the lack of own metabolic activities is probably the most important distinction (whereas symbionts engage in metabolite exchange, for instance).

Excellent point!

Well, GCMS is more for identifying metabolites, as many of the more interesting compounds tend to be volatile (terpenes, flavenoids and lipids come to mind). But isolating them would be difficult endeavor with that, I agree. It really depends on what you want to do, but for a metabolomic approach I would do an extraction and run it on a GCMS to see whether there is anything of interest to begin with (which can be coupled with pre-fractionation). Especially because a decent GCMS is generally way cheaper than a comparable LCMS.

If you need to identify compounds in complex mixtures I do not think there is any better way than LCMS or GCMS. Preparative HPLC can give you very good fractionation, provided that the LC part is set up very accurately and the complexity of your mixture does not overwhelm its separation and fractionation capabilities. Unless there is further purification and prefractionation a complete plant extract would be highly complex, however.

With regards to autoclaving, you can do that. Just be aware that oftentimes autoclaves are not very clean and it may contaminate your solution (although it would be sterile contamination). Depends on the sensitivity of your application. If made cleanly Tris tends to be fairly stable at RT (6 months did not appear to be much of an issue), though I would check pH before use to be certain. Also note that the pH is temperature dependent. Should you put it in the fridge, let it warm up to RT (or whatever the reaction temp is) before use.

Welcome to the wonderful and expensive world of molecular biology. The moment you deal with complex mixtures, the cost skyrocket. With low complexity and well-known compounds you may get away using chromatographic methods with simple detectors.

Now that got incoherent pretty quickly. But in case you wanted to know, bacteria actually produce D-amino acids (e.g. as elements during peptidoglycan synthesis). And as I said, there are enzymes responsible for the interconversion of the amino acid from the D to the L form.

Both amino acids are found in nature, but only L-amino acids are found in proteins. Biosynthesis amino acid can lead to both forms. Transaminase are pretty much unrelated to this question, as they catalyze the exchange between amino and a keto group. Think isomerase.

As BabcockHall mentioned, the regulation is enacted via proteins, not directly via the steroids/hormones (most notably proteins of the nuclear receptor superfamiliy). There are therefore multiple layers that modulate the final expression. One is on the level of the protein with all the possibilities such as by abundance, inhibition, activation, etc. On the metabolite level inactivation of thyroid hormones (I assume we are talking T3/T4 here) there are several pathways but generally is a form of deiodination.

A bit of a tangent, but it isn't it weird to propose to ban information on things that are relatively easy to design even without a detailed manual , whereas purpose-made devices for causing harm (such as guns and ammunition) are freely available? Isn't that a bit putting the cart in front of the horse?

Whether a fragment is sticky or blunt basically depends on how the respective strands are nicked. In most cases star activity changes the sites only by a little (they still tend to be palindromic to some extent, for example) chances are that they will produce same ends as they did under normal circumstances. Due to the loss of specificity the overhang sequence may very well be different, however.

As I mentioned, I do not think that anyone has investigated the precise physics behind it in detail. The best I found was more or less a list of potential interactions with all the usual candidates (with hydrophobic interactions being near the top of the list). There are some models around, mostly from the manufacturers as e.g.: http://www.pall.com/main/oem-materials-and-devices/literature-library-details.page?id=27294.

Just wondering, how is that different from standard optical tweezers (yeah I know, I should read the articles...)?