10 Things Every Molecular Biologist Should Know
Total Page：16
File Type：pdf, Size：1020Kb
10 Things Every Molecular Biologist Should Know Nick Oswald Suzanne Kennedy Megan Hogan Megan Cartwright Edited By Nick Oswald a BitesizeBio.com eBook Preface Molecular Biology is a funny old business... Preface Molecular biology is a funny old One of the core aims of Bitesize Bio website yourself, or if you’d just like to let business. The ton of theoretical (www.bitesizebio.com) is to make it us know what you think (good or bad!) of knowledge that we cram in during our easier to learn on the job as a molecular our efforts, please feel free to get in touch undergrad years is scant preparation biologist by providing a place where via the contact page at BitesizeBio.com. for life at the bench and leaves a lot to these nuggets of vital, often over-looked be said. knowledge can be found. Thanks for reading. When we first hit the lab there are so This short eBook provides 10 such many things to learn before we even nuggets, neatly contained in one place. get started that many things go From How SDS-PAGE works to the unlearned. chemical reason why enzymes have optimal temperatures we hope that there How specific techniques work, what The Bitesize Bio team. will be something in here that will actually is in those kits we use and why enlighten and entertain even the most we use certain approaches rather than experienced scientist. others. If you like the information you find in this Often these nuggets of knowledge can eBook - which is the first of many we’ll mean the difference between be creating on a variety of topics - we understanding where an experiment is would love you pass the file onto your going wrong and not understanding, friends to help spread the word about between getting a result and not getting what we are doing at Bitesize Bio. one. And if you are hungry for more please be But normally, these nuggets can’t be sure to visit us at BitesizeBio.com to get found in lectures or in text books. the latest articles and eBooks we have Instead they must be gleaned through on offer. hard on-the-job experience or passed down by word-of-mouth from more Finally if you have any suggestions for experienced mentors. articles you’d like to see on Bitesize Bio, if you’d like to write something for the ii How SDS-PAGE Works “If you are still wondering why the stacking gel is needed, think of what would happen if you didn’t use one.” Section 01 By Nick Oswald SDS-PAGE (sodium dodecyl sulphate- charge is determined by amino acid charge of the protein. That’s where SDS polyacrylamide gel electrophoresis) is composition i.e. the sum of the positive comes in. commonly used in the lab for the and negative amino acids in the protein separation of proteins based on their and molecular radius by the protein’s The Role of SDS (et al) molecular weight. It’s one of those tertiary structure. techniques that is commonly used but not frequently fully understood. So let’s So in their native state, different proteins SDS is a detergent that is present in the try and fix that. with the same molecular weight would SDS-PAGE sample buffer where, along migrate at different speeds in an with a bit of boiling, and a reducing agent SDS-PAGE separates proteins electrical field depending on their charge (normally DTT or B-ME to break down according to their molecular weight, and 3D shape. protein-protein disulphide bonds), it based on their differential rates of disrupts the tertiary structure of proteins. To separate proteins in an electrical field migration through a sieving matrix (a This brings the folded proteins down to based on their molecular weight, we gel) under the influence of an applied linear molecules. electrical field. need to destroy the tertiary structure by reducing the protein to a linear molecule, Making the rate of protein and somehow mask the intrinsic net migration proportional to molecular weight. The movement of any charged species through an electric field is determined by it’s net charge, it’s molecular radius and the magnitude of the applied field. But the problem with natively folded proteins is that neither their net charge SDS helps to disrupt the tertiary nor their molecular radius are molecular protein structure weight dependent. Instead, their net iv SDS also coats the protein with a The gel matrix be made up at a variety of concentrations uniform negative charge, which masks to produce different pore sizes which, in the intrinsic charges on the R-groups. turn, can produce varying separating SDS binds fairly uniformly to the linear In an applied electrical field, the SDS- conditions that can be adjusted to suit your proteins (around 1.4g SDS/ 1g protein), treated proteins will now move toward needs. meaning that the charge of the protein the cathode at different rates depending is now approximately proportional to it’s on their molecular weight. These The discontinuous buffer molecular weight. different mobilities will be exaggerated system and the stacking gel by the high-friction environment of a gel SDS is also present in the gel to make matrix. sure that once the proteins are To conduct the current through from the linearized and their charges masked, As the name suggests, the gel matrix anode to the cathode through the gel, a they stay that way throughout the run. used for SDS-PAGE is polyacrylamide, buffer is obviously needed. Mostly we use which is a good choice because it is the discontinuous Laemmli buffer system. The dominant factor in determining the chemically inert and, crucially, can easily migration rate of an SDS-coated protein “Discontinuous” simply means that the in a gel is it’s molecular radius. buffer in the gel and the tank are different. SDS-coated proteins have been shown Typically, the system is set up with a to be linear molecules, 18 Angstroms stacking gel at pH 6.8, buffered by Tris- wide and with length proportional to HCl, a running gel buffered to pH 8.8 by their molecular weight, so the molecular Tris-HCl and an electrode buffer at pH 8.3. radius (and hence their mobility in the The stacking gel has a low concentration of gel) is determined by the molecular acrylamide and the running gel has a weight of the protein. Since the SDS- higher concentration capable of retarding coated proteins have the same charge the movement of the proteins. to mass ratio there will be no differential migration based on charge. So what’s with all of those different pH’s? Well, glycine can exist in three different The discontinuous buffer system charge states, positive, neutral or negative v depending on the pH. This is shown in All of the proteins in the gel sample have So the glycine front accelerates past the the diagram below. Control of the an electrophoretic mobility that is proteins, leaving them in the dust. charge state of glycine by the different intermediate between the extreme of the The result is that the proteins are dumped buffers is the key to the whole stacking mobility of the glycine and Cl- so when in a very narrow band at the interface of the gel thing. the two fronts sweep through the stacking and running gels and since the sample-well the proteins are So here’s how the stacking gel works. running gel has an increased acrylamide concentrated into the narrow zone When the power is turned on, the concentration, which slows the movement between the Cl- and glycine fronts. negatively-charged glycine ions in the of the proteins according to their size, the pH 8.3 electrode buffer are forced to And they’re off! separation begins. enter the stacking gel, where the pH is 6.8. In this environment glycine What was all of that about? switches predominantly to the This procession carries on until it hits the zwitterionic (neutrally charged) state. running gel, where the pH switches to If you are still wondering why the stacking This loss of charge causes them to 8.8. At this pH the glycine molecules are gel is needed, think of what would happen move very slowly in the electric field. mostly negatively charged and can if you didn’t use one. migrate much faster than the proteins. The Cl- ions (from Tris-HCl) on the other hand, move much more quickly in Gel wells are around 1cm deep and you the electric field and they form an ion generally need to substantially fill them to front that migrates ahead of the glycine. get enough protein onto the gel. So in the The separation of Cl- from the Tris absence of a stacking gel, your sample counter-ion (which is now moving would sit on top of the running gel, as a towards the cathode) creates a narrow band of up to 1cm deep. zone with a steep voltage gradient that Rather than being lined up together and pulls the glycine along behind it, hitting the running gel together, this would resulting in two narrowly separated mean that the proteins in your sample The effect of buffer pH on glycine fronts of migrating ions; the highly would all enter the running gel at different charge mobile Cl- front, followed by the slower, times, resulting in very smeared bands. mostly neutral glycine front. vi So the stacking gel ensures that all of the proteins arrive at the running gel at the same time so proteins of the same molecular weight will migrate as tight bands.