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Lecture Six: Protein Isolation and Purification 9 [Based on Chapter 3 Berg, Tymoczko & Stryer] (Figures in red are for the 7th Edition) It is possible to separate proteins by differences in their ___________ electric charge DEFINITION pI pI is the Isoelectric Point of a protein. It is the value of pH when the net surface charge is ZERO for that protein Isoelectric Focusing This technique requires a pH gradient gel It uses a gel of polyampholytes Polyampholytes are small multi-charged polymers with different values of pI Applying an electric field to this gel creates the pH gradient Proteins have _________________ of acidic and basic residues They often have an overall positive or negative charge Figure 3-11, page 73 (3-11, page 75) Proteins placed into the gel move within the applied electric field according to their surface charge When the _______________ equals the pI value for any given protein it will cease to move in the field It is isoelectrically focused A ______________ degree of separation can be obtained Figure 3-4, page 70 (3-4, page 72) Ion-exchange Chromatography Ion-exchange chromatography uses beads with a surface charge chemically attached to them This can be a negative (________________ - CM) OR positive (Diethylaminoethyl - DEAE) charge The beads are typically cellulose or agarose Example: Proteins with positive charge will attach to negatively charged beads Other proteins will pass down the column unhindered The positively charged proteins can then be eluted from the column Eluting = ____________ Eluting the bound proteins Add a low concentration of a salt As an example: sodium chloride Sodium ions are clearly very strongly positive The sodium ions ____ to the beads ______ of the proteins Weakly positive proteins will elute off first Increasing the salt concentration will cause more positively charged proteins to elute from the column All the positively charged proteins can be collected as fractions as they come off the column The other way round would work too Positive beads can be used to separate negatively charged proteins Figure 3-7, page 71 (3-7, page 74) Gel Electrophoresis Gel Electrophoresis separates proteins according to their ____ by applying an electric charge through a polymer gel A polyacrylamide gel is almost always used The technique is known as: Polyacrylamide Gel Electrophoresis ==> PAGE Polyacrylamide is chemically inert Figure 3-7b, page 71 (3-7b, page 74) The gel forms as ‘SPAGHETTI-LIKE’ STRANDS The most common form of gel electrophoresis uses: Sodium Dodecyl Sulphate ==> SDS Hence SDS-PAGE SDS is an _________________ It disrupts all non-covalent interactions in proteins SDS binds to amino acid residues in a ratio of around 1:2 All proteins become negatively charged The negative charge on ____________ becomes directly proportional to its mass -Mercaptoethanol is added to disrupt disulphide bonds if there are any present The proteins are now _____________________ Figure 3-7a, page 71 (3-7a, page 74) The protein mixture flows down the gel from the cathode towards the anode Large proteins are impeded in the gel by the strands The smaller proteins pass easily between the strands Summarise: Smallest proteins fastest through the Strands Figure 3-9, page 73 (3-9, page 75) The protein separation provides a direct measurement of their ________ Proteins with known molecular masses are run as a scale marker beside those with unknown mass Differences in mass of ~2% between proteins can be determined using SDS-PAGE Around 10 residues difference Affinity Chromatography Affinity Chromatography makes use of the fact that many proteins tightly bind small specific molecules as part of their function Figure 3-5, page 70 (3-5, page 72) Example: Concanavalin A Concanavalin A binds glucose very tightly It is possible to covalently attach glucose to beads in a column Pass the crude protein mixture containing Concanavalin A down the column The Concanavalin A will bind to the glucose on the beads All remaining proteins will pass through unhindered Concanavalin A can then be removed by passing a concentrated solution of glucose down the column Concanavalin A binds better to the ‘free’ glucose than to the ‘bound’ glucose on the beads The Concanavalin A will elute from the column bound to the ‘free’ glucose Typically the ‘free’ glucose would be removed by ________ General Technique A protein, Y, recognises and binds a small molecule, X Covalently attach X to beads and place these in a column In a mixture of proteins containing Y only that protein will bind to X on the beads and will be retained by the column The rest of the proteins will pass on through the column Passing down the column a concentrated solution of ‘free’ X will remove protein Y from the column Protein Y will be removed as pure protein Summary of Lecture Six: Proteins can be separated due to differences in their surface electric charge Isoelectric Focusing On a pH gradient gel with an applied electric field, a protein will move within the field until the charge on a protein is zero This is its Isoelectric Point (pI) Proteins can be separated this way Ion-exchange Chromatography Beads on a column with a given attached surface charge will bind proteins with an opposing charge The bound proteins are then separated and removed by the addition of a gradually increasing concentration of a salt Gel Electrophoresis Most common form of the technique is: SDS PAGE Separates proteins both by size and charge This technique can measure a proteins mass Affinity Chromatography Separates proteins with specific binding for a small molecule attached to beads on a column The protein is eluted off by passing a concentrated solution of the ‘free’ small molecule down the column