SDS PAGE Lecture PDF

BENG0004 Biochemistry and Molecular Biology Dr. Michael Thomas Techniques in protein analysis SDS PAGE  SDS Polyacrylamide gel electrophoresis Being able to separate out a solubilised, heterogeneous mix of proteins is an essential technique in the study of prot...

BENG0004 Biochemistry and Molecular Biology Dr. Michael Thomas Techniques in protein analysis SDS PAGE  SDS Polyacrylamide gel electrophoresis Being able to separate out a solubilised, heterogeneous mix of proteins is an essential technique in the study of proteins The most common technique in biochemistry for this is sodium dodecyl sulphate, polyacrylamide gel electrophoresis – a.k.a. SDS-PAGE - separation through size and “charge” Remember, the overall electrical charge of DNA is negative (due to the negatively charged phosphate group on the ribose sugar backbone), hence separation of DNA fragments is relatively simple using a gel matrix (agarose) and an electrophoretic system. However, since proteins vary in their overall net charge, separating a mixture of solubilised proteins is more complicated. We can still use a gel matrix, but the properties of the gel and the overall conditions of electrophoresis (e.g. buffers) are quite different  SDS PAGE Proteins don’t have a uniform charge and so won’t all migrate in an electric field in the same direction (which is actually useful! – Omics lecture). If all proteins can be given an uniform charge based on their size (length) we can then separate them according to size in electrophoresis. The detergent SDS (sodium dodecyl sulphate) is used to denature (unfold and unwind) proteins and bind to the proteins in a uniform amount depending on their size. The SDS coats the protein chains and gives them a net negative charge proportional to their size. This also means that any multi-subunit proteins are completely separated. We must also add powerful reducing agents such as DTT or mercaptoethanol to reduce any disulphide bonds holding two proteins together of parts of a protein chain.  Sample preparation prior to SDS-PAGE It is normal to heat to close to 100°C to completely denature proteins with SDS and DTT Presence of reducing agents (DTT) also breaks disulphide linkages We now have proteins uniformly denatured and with a constant amount of SDS per unit length. The sample can now undergo electrophoresis.  The gel – polyacrylamide Polyacrylamide is a very hydrophilic polymer which is The buffer used to also tough. dissolve the monomers for It needs the polymer chains to polymerisation has be cross linked to one SDS in it so that the another (using methylene proteins remain bisacrylamide) to form pores denatured for the sieving effect needed throughout the in gel electrophoresis. electrophoresis. Tetramethylethylenediamine (TEMED) and an initiator which donates free electrons, We can alter the pore (ammonium persulfate) are size and thus the size used to catalyse the range of proteins polymerisation reaction separated by using different amounts of Forms the basis of the cross linker bis- the gel matrix acrylamide.  The gel – Polyacrylamide (PA) Polyacrylamide gels are actually gels made up of at least two different densities of PA (see later) so are classed as discontinuous gels (agarose gels are continuous) Because of this, they are actually quite hard (well, fiddly) to make Nowadays, most people buy “precast” gels with a pre-specified PA density (roughly £10 per gel) There are a lot of variations in the type of gel as well (again, see later)  Discontinuous gel systems in SDS PAGE Agarose gels are the same concentration Protein gels are made of two parts (stacking gel and of agarose throughout (continuous). The separating gel) which contain different buffers are also the same. concentrations of PA and therefore have different pore sizes. The two gels sections are also made using different buffers (e.g. Tris-Glycine and Tris-Cl) – hence the buffer and gel system are discontinuous. Stacking gel: upper part of the gel that contains a lower concentration of acrylamide (4-6%) in order to “sieve” proteins and concentrate them into a sharp band before entering the separating portion on the gel (this gives neater bands/greater resolution). Separating (resolving) gel: this is where the proteins undergo bulk separation through size. Low concentration acrylamide and bis-acrylamide polymerization yields larger pores for high molecular weight separation (see “protein migration” later). Thanks to the presence of SDS, all the loaded protein has a negative charge thus flows to the anode…  Electrophoresis in SDS PAGE There are two buffer containing chambers in a protein gel system – one to fuel the electrophoretic process in the stacking gel and the other for the resolving gel. Since the gels are discontinuous and the buffers used to make the two separate parts of the gels are different, there is some different electrochemistry going on Essentially: Buffer in the upper chamber forces proteins into the gel (stacking) Buffer in the lower chamber forces proteins through the gel (resolving  Protein compressed between glycinate & chloride Chloride ions are green, glycinate are orange and denatured proteins are blue. When the ionic front reaches the resolving gel (pH of 8.3), the glycinate and chloride mobility increases. The increase in pH increases the mobility of the proteins as well. The pore size is smaller in the resolving gel and starts to separate the proteins according to size, retarding the large ones more than the smaller ones. The mobility of the various components is now Stacking chloride > glycinate > protein gel pH 6.7 Due to the SDS, the proteins have a constant charge to Resolving mass ratio and become separated according to size. gel pH The separation is a linear relationship between the log 8.3 of the molecular weight and the electrophoretic mobility (or distance travelled from the origin) A bit of history The Tris-glycine-chloride buffer system was developed by Prof Ulrich Laemmli at the MRC in Cambridge and his paper describing this buffer system became the second most highly cited papers of all time. Nature 227, 680 - 685 (1970) Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 Graph depicting the linear relationship between molecular weight mobility (RF value - the distance travelled by a given component divided by the distance travelled by the solvent front). Biochemistry, Berg et al, 8th edition. Pages 72+73.