Lecture 05: Chromatography and Electrophoresis PDF

Unit III : Chromatography and Electrophoresis Biological and Biochemical Techniques BBT-304 Dr. Subhash Kudale Electrophoresis Lecture V : IEF and 2-D Electrophoresis Isoelectric focusing Electrophoresis - Isoelect...

Unit III : Chromatography and Electrophoresis Biological and Biochemical Techniques BBT-304 Dr. Subhash Kudale Electrophoresis Lecture V : IEF and 2-D Electrophoresis Isoelectric focusing Electrophoresis - Isoelectric Focusing – 3 Lecture V – IEF electrophoresis Isoelectric focusing Electrophoresis - Isoelectric Focusing – It is ideal for separation of amphoteric substance such as proteins because it is based on the separation of molecules according to their different iso- electric points. It has high resolution, being able to separate proteins that differ in their iso-electric points by as little as 0.01 of a pH unit. Most widely used system for IEF utilizes horizontal gels on glass plates or plastic sheets. Separation is achieved by applying a potential difference across the gel that contains a pH gradient, which is formed by the introduction of the ampholytes (complex mixtures of synthetic polyamino-polycarboxylic acids). Ampholytes are available in different pH ranges (pH 3 to 10) and a pH range is chosen such that the sample being separates will have their isoelctric point within this range. 4 Lecture V – IEF electrophoresis Isoelectric focusing Electrophoresis - Isoelectric Focusing – Commercially available ampholytes includes bio-lyte and pharmalyte. Traditionally, 1-2mm thick isoelectric focusing gel have been used, but nowadays, thin layer IEF gels of only 0.15mm thick, are used, prepared using a layer of electrical insulation tape as the spacers between the gel plates. Since, this method requires the proteins to move freely according to their charge under the electric field. To avoid any sieving effect within the gel, IEF is employed in low percentage gels. Commonly, 4% polyacrylamide gel are used but agarose is used, for the study of high relative molecular mass proteins that may undergo some sieving even in a low percentage acrylamide gel. Thin layer IEF gel is prepared by mixing ampholytes, gel material and riboflavin and the mixture is then poured over a glass plate (25cmX10cm). 5 Lecture V – IEF electrophoresis Isoelectric focusing Electrophoresis - Isoelectric Focusing – The second glass plate is then placed on top of the first to form gel cassette and the gel polymerization is carried out by photo polymerization by placing the gel in front of a bright light. This takes 2-3hrs. After the gel is set, the glass plates are removed apart to reveal the gel stuck to one of the glass sheets. Electrode wicks (3mm) which are strips of wetted filter paper (anode- phosphoric acid and cathode-sodium hydroxide) are laid along the long length of each side of the gel and a potential difference is applied. Under the effect of this potential difference, the ampholytes forms a pH gradient between anode and a cathode. The power is then turned off and samples is applied by laying on the gel small squares of filter paper soaked in the sample. 6 Lecture V – IEF electrophoresis Isoelectric focusing Electrophoresis - Isoelectric Focusing – A voltage is again applied for about 30min to allow the sample to electrophorese off the paper into the gel, at which time the paper squares can be removed from the gel. Depending on which point on the pH gradient, the sample has been loaded, proteins that are initially at pH regions below their isoelectric point will be +vely charged and migrate towards cathode. As they proceed, the surrounding pH will increase steadily and the +ve charge on protein will decrease correspondingly until eventually the protein arrives at Zwitter ion form with no net charge, hence the further movement ceases. Similarly, the proteins at pH region above their iso-electric point will be -vely charged and will migrate towards the anode until they reach their iso-electric points and become stationary. To achieve rapid separation (2-3hrs) relatively high voltage (up to 2500V) are used. 7 Lecture V – IEF electrophoresis Isoelectric focusing Electrophoresis - Isoelectric Focusing – Following electrophoresis, the gel must be stained to detect the proteins, as this can not be done directly, because the ampholytes will stain too, giving totally blue gel. So, for this purpose, the gel is therefore washed with fixing solution (10% v/v trichloroacetic acid), which precipitates the proteins and allows much smaller ampholytes to be washed out. The gel is then stained with Coomassie brilliant blue and then destained. The pI of a particular protein may be determined conveniently by running a mixture of proteins of known isoelectric point on the same gel. After staining, the distance of each band from the electrode is measured, and a graph of distance for each protein against its pI is plotted and by this calibration line, the pI of unknown protein can be determined. 8 Lecture V – IEF electrophoresis 2–D Gel Electrophoresis - 2-D Gel Electrophoresis - 9 Lecture V – 2-D Gel electrophoresis 2–D Gel Electrophoresis - 2-D Gel Electrophoresis – It combines the techniques of IEF, which separates proteins in a mixture according to charge (pI) with the size separation technique of SDS-PAGE. When combined to give two-dimensional PAGE, the most sophisticated analytical method for separating proteins available is obtained. The first dimension (IEF) is carried out in polyacrylamide gels in narrow tubes (Internal diameter 1-2mm) in presence of ampholytes, 8M urea and non-ionic detergent. The gel is then extruded from the tube by applying slight pressure to one end, incubated for 15mins in a buffer containing SDS, then placed along the stacking gel of an SDS -added gel and fixed in a place by pouring molten agarose, in electrophoresis buffer over the gel, Once the agarose has set, electrophoresis is commenced and the SDS bound proteins run into gel and are separated according to size. 10 Lecture V – 2-D Gel electrophoresis Thank You