Coomassie Brilliant Blue Dye - the science of SDS-PAGE staining скачать в хорошем качестве

Coomassie Brilliant Blue Dye - the science of SDS-PAGE staining

You can think of the SDS-PAGE gel’s matrix as maze with protein “treasure” hidden throughout. Unlike the proteins, with moved straight down the gel because the electric field was compelling them that way, the dyeing step happens after you turn off the electricity and transfer your gel from its plate sandwich to a staining box. Here, there’s no directionality to movement of things, so the dye will move wherever the heck it wants! (though the protein will be stuck in place through chemical-induced clumping as we’ll see).  blog form: http://bit.ly/cbbgelstaining   The dye travels randomly through the gel maze by diffusion (the molecules move around randomly, ricocheting off the things they run into with the NET RESULT that they move from areas of high concentration to areas of low concentration). When it finds protein treasure, it latches on. And since the dye’s blue it tells us where the protein is.    But it’s really hard to find treasure that’s trying to escape! So we need to get the treasure to stay put – the FIXATION step uses an alcohol and/or acid to make the protein to precipitate (clump up) so it gets stuck in place so our treasure-hunting dye can find it. We also have to watch out for overly-eager treasure hunters that latch onto “fool’s gold” (stain the gel itself) causing high background. This leads to an overall treasure-hunting scheme of:    1. GEL ELECTROPHORESIS – separate proteins by size by unfolding them, coating them in negatively-charged detergent (SDS) & using that negative charge to motivate them to travel through a polyacrylamide gel mesh towards a positive charge. The bigger (longer) proteins get tangled up more, so they travel slower and have progressed less (so higher up on gel) when you turn off the power. More here: http://bit.ly/sdspageruler 2. WASH – remove free SDS, etc.   3. FIX – trap the treasure – the gel has to allow proteins to move when you want them to, but not too easily – Ideally they’d only move when power’s on, but molecules like to move and if they can they will – so (especially the small ones) can start wandering off even when the power’s off). To prevent that wandering, you add an alcohol and/or acid to get them to clump and get stuck in place.   4. STAIN – send in the treasure hunters – stick the gel in a bath of dye – the dye enters, latches onto the protein and gets stuck too (this step’s often combined w/the fixing step)  5. DESTAIN – call off the hunt – get the treasure-less treasure hunters to leave so you can better see where the treasure-full ones are (some rapid stains have low backgrounds & you just destain in water, letting the dye diffuse out, but some methods use more complex destaining)    There are lots of different formulations including “Classic Coomassie” which has really eager treasure-hunters that can find tiny amounts of treasure but are also fool’s-gold-happy – it’s super sensitive and eventually gives you nice crisp bands, but takes a lot of destaining to reveal them. Alternative “Colloidal Coomassie” recipes are becoming more common because they’re faster & more eco-friendly – these forms keep groups of treasure-hunters hanging out outside the gel & gradually send them in until all the treasure’s found and bound.    There are 2 main forms, R-250 & G-250. Both are blue, but R’s more “reddish” & G’s more “greenish” (although the color depends on the pH and whether and what it’s bound to which is why it can be used to measure protein concentrations in something called a “Bradford assay” http://bit.ly/bradforduv ). “250” was originally a purity/strength indicator. R-250 is more sensitive , but G-250 can be made into forms that produce lower background, with faster protocols.    We get the G-250-based “quick stain” we use most of the time pre-made, but we make our own “Classic” R-250 stain. It’s a really simple recipe -  only 4 ingredients: water, acetic acid (AcOH), methanol (MeOH), & CBB - but it takes a while to prepare… more here: http://bit.ly/2QJNwLy    CBB has sulfuric acid groups that can be negative or neutral depending on pH. Under the conditions of the staining solution it has overall ➖charge (anionic), so it binds (reversibly) to ➕-charged parts of proteins (basic amino acids like Arg, Lys, & His) through electrostatic interactions (opposites attracting). note: those side chains aren’t always positive, but we stain the gel in acidic conditions, where they’re more likely to be –   CBB also binds to non-charged protein parts (especially the ring-y (aka aromatic) amino acids like Phe, Tyr, & Trp) “generically” through “Van der waals” interactions, which involve shifting around of electrons when molecules get close together.  These interactions are individually weak but they add up (they’re what allow geckos to walk up walls!). Proteins with unusually high proportions of ring-y amino acids tend to stain better. An example is BSA (bovine serum albumin)