Lecture 3 (SDS PAGE) PDF

Principles and Techniques of SDS- PAGE LECTURE Farah Haziqah3Meor (BOE201) Termizi, PhD G08, 276, School of Biological Sciences, USM [email protected] ATTENDANCE – L...

Principles and Techniques of SDS- PAGE LECTURE Farah Haziqah3Meor (BOE201) Termizi, PhD G08, 276, School of Biological Sciences, USM [email protected] ATTENDANCE – LECTURE 3  What is protein electrophoresis? DNA are negatively charged molecules due to their sugar- phosphate backbone. When electrophoresed, they automatically migrate towards the positive electrode. In contrast, a protein molecule may carry an overall positive or negative charge or may be uncharged depending on its amino acid sequence. Further, amino acids, due to having both NH2+ and COO- groups and consequently the protein molecule, may have variable overall charge as the pH varies.  What is protein electrophoresis? So, if you think about it, when a mixture of proteins, at a certain pH, are put in an electric field for electrophoresis, depending on their overall charge, some of the protein molecules may migrate towards the cathode, others towards the anode, and some may not migrate at all. To overcome this uncertainty, and to ensure that all proteins have an overall similar charge and migrate towards one electrode, a special treatment is done to the protein mixture before electrophoresis. The protein sample mix is treated with the detergent SDS (Sodium Dodecyl Sulfate). SDS-PAGE - Sodium dodecyl sulfate-polyacrylamide gel electrophoresis A technique used for the separation of proteins based on their molecular weights. The molecular weight of a protein is the sum of the masses of all its constituent amino acids. To better understand, let’s look at little closer at each of these terms. SDS stands for Sodium Dodecyl Sulfate, a detergent. PAGE is the abbreviation of Poly Acrylamide Gel Electrophoresis. = And this is where we get the combined term SDS-PAGE. Electrophoresis is a procedure that relies on an electric current to separate macromolecules, specifically in this case, proteins in a mixture. The separation here is solely based on the protein’s molecular weights. Polyacrylamide, is a polymer that the gel is made up of. Applications SDS-PAGE Protein Purification: Assessing the purity of protein samples. Molecular Weight Estimation: Determining the molecular weight of proteins. Protein Identification: Identifying proteins in complex mixtures. Comparative Analysis: Comparing protein expression levels under different conditions Outline of recombinant protein production and purification in Escherichia coli.  For this different protein molecules with different shapes and sizes, needs to be denatured (done with the SUMMAR Y aid of SDS) so that the proteins lose their secondary, tertiary or quaternary structure. The proteins being covered by SDS are negatively charged and when loaded onto a gel and placed in an electric field, it will migrate towards the anode (positively charged electrode) are separated by a molecular sieving effect based on size. After the visualization by a staining (protein- specific) technique, the size of a protein can be calculated by comparing its migration distance with that of a known molecular weight ladder (marker).  Unlike DNA and RNA, proteins vary in charge according to the amino acids incorporated, which can influence how they run. Amino acid strings may also form secondary structures that impact their apparent size and consequently how they are able to move through the pores. It may therefore sometimes be desirable to denature proteins prior to electrophoresis to linearize them if a more accurate estimate of size is required.  The pores formed in polyacrylamide are smaller than those of agarose, used for agarose gel electrophoresis. This makes it more suitable for the separation of proteins over large polynucleotide DNA or RNA fragments and allows the separation of relatively small proteins. Consequently, when referring to “protein electrophoresis”, the separation technique will most often be referring to is PAGE.  Preparing proteins for SDS-PAGE The anionic detergent, sodium dodecyl sulphate (SDS), in combination with heat and sometimes a reducing agent is used to denature proteins prior to electrophoretic separation in a process known as SDS-PAGE. The heat disrupts the hydrogen bonds that hold secondary and tertiary structures while a reducing agent, such as β-mercaptoethanol (BME), cleaves disulfide bridges. Heating The three-dimensional shape of a protein molecule is largely governed by hydrogen bonds. For SDS-PAGE, since protein samples need to be linearized into their primary structures, these hydrogen bonds need to be broken. For this, as part of preparation for SDS-PAGE, the sample is heated for a few minutes at around 95°C. Heating destroys hydrogen bonds.  Proteins are linearized and complex with the SDS so that all have a similar mass-to- charge ratio. This eliminates the influence of structure and charge, and proteins are separated solely on the basis of differences in their molecular weight. MATERIALS REQUIRED FOR SDS-PAGE Vertical Electrophoresis System  This system was developed by Ulrich K. Laemmli and is typically used to separate proteins of 5 - 250 kDa. Polyacrylamide gels  Polymerized acrylamide (polyacrylamide) forms a mesh-like matrix suitable for the separation of proteins of typical size. The strength of the gel allows easy handling. Preparation of Polyacrylamide Gel  The process of making a gel is called casting. The gel Generally, for protein electrophoresis, the same polyacrylamide gel has two parts, a stacking part and a resolving gel. A resolving gel solution (acrylamide ~10%, pH ~8.8) forms the bottom of the casting tray, and so it is poured before the stacking gel. Tetramethylethylenediamine (TEMED) Ammonium persulfate (APS) Isopropanol is then layered over the resolving gel before it solidifies - prevents air bubbles and protects the resolving gel from drying out. Also, this helps in flattening the top layer of the resolving gel rather than getting curvy.  The stacking gel, which has a lower acrylamide percentage (~4%), a different pH (~6.8) and ionic composition than the resolving portion, is then poured into the casting tray. After pouring the stacking gel mix, a comb is placed on top to create “wells”. Once the gel is ready, the protein samples are added into these wells for the electrophoretic separation to start. Run the SDS-PAGE The Buffer The running apparatus has electrodes connected to a source of electricity and is filled with a running buffer solution. This buffer, due to its ions, facilitates the flow of electric current – the driving force behind electrophoretic separation. They serve to maintain a certain pH; for example, around 6.8 and 8.8 for stacking and resolving gels respectively. In SDS-PAGE, three (3) different buffers are used: 1) for casting the gel 2) for sample preparation and loading 3) for running the gel Loading samples into the wells of gel An SDS-PAGE loading buffer contains SDS & BME (necessary to denature the proteins in the sample) and also contains glycerol and a dye, such as bromophenol blue. Glycerol - Increases its density to ensures that the sample drops to the bottom of the well during gel loading. - This prevents spillover and contamination of samples between the wells which may produce erroneous experimental results. Bromophenol blue - For tracking purposes to monitor how far the samples have migrated in the gel. - Ensure that electrophoresis is stopped at the proper time so that proper separation is obtained. - Ensure that the samples do not run all the way out of the gel. - Dye migrates towards the positive electrode with the SDS-covered protein samples. *Bromophenol blue is negatively charged at the same pH at which SDS- PAGE is done. Protein ladder One of the wells in the gel is loaded with a protein ladder. The ladder is an assortment of multiple highly purified polypeptides of predetermined and varying sizes. These polypeptides, due to their sizes differing between each another, migrate different known distances in the gel during electrophoresis. Each rung of the ladder corresponds to a polypeptide of a certain size (molecular weight). Visualization of the gel Visualization of protein bands is carried out by incubating the gel with a staining solution. The two most used methods are Coomassie-blue staining and silver staining. Silver staining is a more sensitive staining method than Coomassie staining and is able to detect 2–5 ng protein per band on a gel. Many protocols are available but in order to increase reproducibility, use of a commercially available kit is recommended. A. Coomassie Blue staining Stain the SDS-PAGE Gel (Coommasie-blue Staining) A. Silver staining Interpreting Results from a Protein Gel