Housekeeping genes/proteins, internal references/loading controls & related terms: constitutive etc. скачать в хорошем качестве

Housekeeping genes/proteins, internal references/loading controls & related terms: constitutive etc.

“All” cells in your body have the ability to make every protein you’d ever need. But just like you don’t need snow boots in the summer, and you never need them in Southern California, your cells don’t need all those proteins all the time, and different cell types have different needs that can change depending on the time and environment. Bottom line, the levels of most of these proteins will vary between cells and under different conditions. We can say they’re differentially expressed (more on this all in a second). And if we measure this expression (such as with qPCR or western blots) we can learn about what’s going on in the cells. But in order to fairly compare expression between samples we need to normalize them - remove any bias that comes from having a greater total starting amount of sample. Otherwise you won’t know, for instance, if you’re seeing a stronger signal because the protein you’re looking for really is expressed more or if you just started with more cells. In order to do this normalization, we often turn to housekeeping genes/proteins to serve as internal references or loading controls. blog form: https://bit.ly/housekeepinggenes Housekeeping genes/proteins are more like underwear than snowboots. They’re something you’re always needing and thus always making no matter where you are or what time it is (showers and nudists excluded for the sake of the analogy). Common “cellular underwear” we use as housekeeping genes/proteins are GAPDH (an enzyme involved in helping break down sugar in glycolysis), actin, & tubulin (components of the cytoskeleton - a network of tubes and stuff that gives cells their shape and moves stuff around in them). In jargon terms we can describe housekeeping genes as things that are constitutively and ubiquitously expressed. Constitutive refers to something that’s going on "all the time" without needing to be told to ("no" regulation of activation). And ubiquitous refers to it happening "everywhere" ("all" cell types). We can use these terms in various contexts. For example we can talk about some enzymes and receptors having constitutive activity if they don’t need to be stimulated in order to be active). But, for housekeeping genes/proteins, the “something happening” that we are referring to is gene expression. Basically the genes getting “used” (transcribed into RNA which can then be translated into protein). Much much more on gene expression as well as how we can measure it in other posts, especially: https://bit.ly/expression_measurement; YouTube:    • Gene expression - levels of regulation & m...      Since this is a more technical post, I’m going to assume people have a grasp of the basic central dogma - that is: 1. the permanent recipes for making proteins are written in the form of segments of DNA called genes 2. Messenger RNA (mRNA) copies of those recipes get made in a process called transcription 3. Protein gets made following those recipes in a process called translation There are various forms of regulation that can occur at each of these steps, which allows cells to control what they make, how much they make, and when they make it. This lets your cells differentiate themselves from other cell types and respond to changing needs. By measuring the levels of mRNA transcripts (such as with qPCR) and/or protein (such as with western blots) we can get a glimpse at how highly various genes of interest are being expressed. And the real power comes from being able to compare this expression under different conditions. But that power relies on a way to normalize your results. Enter the housekeeping genes/proteins. More details in the video, but basically you measure levels of a housekeeping gene or protein (e.g. tubulin, actin) in the same sample as the thing you really care about, then divide by the relative amount of the housekeeping protein to control for differences in how much sample you started with. So, for example, to normalize western blots using housekeeping proteins as loading controls, the basic procedure is: 1. quantify band intensities (such as with imageJ or similar) 2. find the lane with the most housekeeping protein 3. divide the amount of housekeeping protein in each lane by that highest amount to get the lane normalization factor (LNF) 4. divide the band intensity for the protein of interest in each lane by that lane's LNF If you’re wondering how you can probe for different things on the same blot, fear not! I have a whole post on that: blog form: https://bit.ly/western_reprobe ; YouTube:    • Probing a western blot membrane for multip...   But your basic options are multiplexing, using conjugated primaries (which you can often get cheaply for common housekeeping proteins), strip and reprobe, or cut and probe separately. Finished in comments