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BT-202 Netaji Subhas Institute of Technology, Dwarka, New Delhi. Dr. Amita Pandey Aug 24, 2011
Syllabus for mid-term Properties of water  ✔ Acids and bases and buffers  ✔ Covalent bond ✔ Non-covalent interactions in b...
Learning check! <ul><li>What is the molecular formula for glucose? </li></ul><ul><li>C6 H12 O6 </li></ul><ul><li>CO2 </li>...
<ul><li>Maltose is composed of which two simple sugars? </li></ul><ul><li>Two fructose </li></ul><ul><li>Glucose and galac...
Polysaccharides <ul><li>Polysaccharides are polymers of monosaccharides also called glycans.  </li></ul><ul><li>Polysaccha...
 
<ul><li>Starch in plants and glycogen in animals are the most important storage polysaccharide. </li></ul><ul><li>Both occ...
Starch and Glycogen
Starch <ul><li>Contains amylose and amylopectin which are glucose polymers. </li></ul><ul><li>Amylose has long chains of D...
<ul><li>Glycogen is more extensively branched (branch on average at every 8-12 residues). More compact than starch. </li><...
Homoploysaccharides as structural biomolecules <ul><li>Cellulose </li></ul><ul><li>Cellulose is fibrous, tough, and water ...
Cellulose <ul><li>Termites, bacteria, ruminants, cattles, and wood fungus can break down  </li></ul><ul><li>cellulose. </l...
Chitin <ul><li>Composed of  N -acetylglucosamine residues in ( β 1  4)-linkage. </li></ul>
Heteropolysaccharides as structural components <ul><li>Glycosaminoglycans </li></ul><ul><li>repeating disaccharide units  ...
<ul><li>Present in ECM. </li></ul><ul><li>Hyaluronan </li></ul><ul><li>Chondritin sulfate </li></ul><ul><li>Keratan sulfat...
<ul><li>Peptidoglycan </li></ul><ul><li>Alternating </li></ul><ul><li>( β 1  4)-linked </li></ul><ul><li>N -acetylglucosam...
Polysaccharides as information carriers <ul><li>Communication between cell and extracellular components. </li></ul><ul><li...
Glycoconjugates <ul><li>Proteoglycans </li></ul><ul><li>Glycoproteins </li></ul><ul><li>Glycolipids </li></ul>
Glycoproteins <ul><li>Carbohydrate protein conjugates.  </li></ul>
Glycoproteins <ul><li>Eg. Mucins, glycophorin A, immunoglobins, and certain harmones, collagen. </li></ul>Oligosaccharide ...
<ul><li>Glycomics   </li></ul><ul><li>systematic characterization of all the carbohydrate component of the cell or tissue,...
<ul><li>What is the biological advantage of adding oligosaccharides to proteins? </li></ul><ul><li>How Lysozyme kills bact...
Separation techniques for Carbohydrates
Chromatography <ul><li>Mobile phase </li></ul><ul><ul><li>Mixture dissolved in liquid or solid </li></ul></ul><ul><li>Stat...
Gel Filteration <ul><li>Gel-filtration </li></ul><ul><ul><li>Size/molecular exclusion chromatography </li></ul></ul><ul><u...
Affinity chromatography <ul><li>Affinity </li></ul><ul><ul><li>Matrix chemically altered to include a molecule designed to...
Affinity Chromatography <ul><li>Separation is based on highly specific interactions. </li></ul><ul><li>Lectin affinity chr...
High Performance Liquid Chromatography (HPLC) <ul><li>-Stationary phase = small uniform particles, large surface area </li...
Mass Spectrometery <ul><li>Determines the mass-to-charge ratio of charged molecules. </li></ul><ul><li>loaded sample is va...
Oligosaccharides in a group of glycoproteins analyzed  by MALDI MS
Learning check! <ul><li>Heparin is routinely added to blood samples obtained for clinical analysis to </li></ul><ul><li>in...
<ul><li>Hyaluronidase is an enzyme founf in pathogenic bacteria which hydrolysis </li></ul><ul><li>Chondritin sulfate </li...
Amino Acids, Peptides, and Proteins
Amino acids <ul><li>The building blocks of proteins </li></ul><ul><ul><li>Essential amino acids </li></ul></ul><ul><li>20 ...
Zwitterions <ul><li>Both the –NH 2  and the –COOH groups in an amino acid undergo ionization in water. </li></ul><ul><li>A...
Zwitterions <ul><li>Soluble in polar solvents due to ionic character </li></ul><ul><li>Structure of R also influence solub...
Classification of Amino Acids <ul><li>Classify by structure of R </li></ul><ul><ul><li>Nonpolar </li></ul></ul><ul><ul><li...
Non-polar, aliphatic R groups
Polar, uncharged R group
Disulfide Bonds
Aromatic R groups
UV-Vis Spectroscopy <ul><li>Absorbance used to monitor protein concentrations </li></ul>
Acidic and Basic Amino Acids <ul><li>Acidic  </li></ul><ul><ul><li>R group = carboxylic acid  </li></ul></ul><ul><ul><li>N...
Uncommon amino acids <ul><li>4-hydroxyproline </li></ul><ul><li>5-hydroxylysine </li></ul><ul><li>6-N-methyllysine </li></...
Acid-base Properties <ul><li>AAs also have multiple pK a ’s due to multiple ionizable groups </li></ul>pK 1  ~ 2.2 (proton...
3-letter and 1-letter abbreviations
pH and Ionization
Titration of Glycine <ul><li>pK 1 </li></ul><ul><ul><li>[cation] = [zwitterion] </li></ul></ul><ul><li>pK 2 </li></ul><ul>...
Titration curve of histidine -Aas with ionizable R groups have three pKa  values
pI  <ul><li>For AAs with 3 pK a ’s, pI = average of two relevant pK a  values </li></ul><ul><li>Consider lysine (pK 1  = 2...
Stereochemistry of AAs <ul><li>All amino acids (except glycine) are optically active </li></ul><ul><li>Fischer projections...
D and L Configurations  <ul><li>d  = dextrorotatory   </li></ul><ul><li>l  = levorotatory </li></ul><ul><li>D, L = relativ...
Importance of Stereochemistry <ul><li>All AA ’s found in proteins are L geometry </li></ul><ul><ul><li>S  enantiomer for a...
Learning Check! <ul><li>Glycine side chain is –CH3 </li></ul><ul><li>Alanine side chain is CH2-phenyl </li></ul><ul><li>Ph...
<ul><li>Give the single letter notation for any one of the polar neutral amino acids. </li></ul><ul><li>S, T, C, N, Q </li...
The Peptide Bond
Polypeptides <ul><li>Linear polymers (no branches) </li></ul><ul><li>Terminal residues: </li></ul><ul><ul><li>Free amino g...
Naming  Peptides <ul><li>Name from the free amine (NH 3 + ) </li></ul><ul><li>Use - yl  endings for the names of the amino...
Learning Check! Ser-Gly-Tyr-Ala-Leu serylglycyltyrosylalanylleucine
Calculate number of AAs present in a protein <ul><li>For a simple protein  </li></ul><ul><li>Molecular weight / 110 </li><...
Separation and purification <ul><li>Determine a source (tissue) </li></ul><ul><li>Extract protein </li></ul><ul><ul><li>Su...
Concentration of salts <ul><li>Salting out </li></ul><ul><ul><li>Continue to increase [salt] decreases [protein] </li></ul...
Types of Chromatography <ul><li>Paper </li></ul><ul><ul><li>Stationary phase = filter paper </li></ul></ul><ul><ul><li>Sam...
Types of Chromatography <ul><li>Ion-exchange chromatography </li></ul><ul><li>Gel filtration / size-exclusion chromatograp...
Electrophoresis Separation of proteins based on their charge in an electric field.
<ul><li>-binds to proteins and gives them  </li></ul><ul><li>a similar charge-to-mass ratio. </li></ul><ul><li>-Unfolds th...
Isoelectric focusing <ul><li>-determining isoelectric point of a protein. </li></ul><ul><li>-Gradient gel is prepared by a...
<ul><li>-2D electrophoresis </li></ul><ul><li>-separation of proteins  </li></ul><ul><li>With identical molecular </li></u...
<ul><li>E-mail address of one student required. </li></ul><ul><li>Electronic submission of assignments. </li></ul>
Agar <ul><li>Present in cell walls of marine algae and sea weeds. </li></ul><ul><li>D-galactose and an L-galactose derivat...
 
 
Ion Exchange Chromatography <ul><li>Ion-exchange </li></ul><ul><ul><li>Stationary phase = chemically modified to include c...
Ion Exchange Chromatography
Affinity Chromatography
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  1. 1. BT-202 Netaji Subhas Institute of Technology, Dwarka, New Delhi. Dr. Amita Pandey Aug 24, 2011
  2. 2. Syllabus for mid-term Properties of water ✔ Acids and bases and buffers ✔ Covalent bond ✔ Non-covalent interactions in biological system ✔ Carbohydrates ✔ Proteins Enzymes Lipids Nucleic acids Vitamins and Co-enzymes Separation technique for biomolecules
  3. 3. Learning check! <ul><li>What is the molecular formula for glucose? </li></ul><ul><li>C6 H12 O6 </li></ul><ul><li>CO2 </li></ul><ul><li>C12 H22 O11 </li></ul><ul><li>C12 H22 O11 </li></ul><ul><li>Which of the following is a simple sugar or monosaccharide? </li></ul><ul><li>galactose </li></ul><ul><li>Sucrose </li></ul><ul><li>maltose </li></ul><ul><li>lactose </li></ul>
  4. 4. <ul><li>Maltose is composed of which two simple sugars? </li></ul><ul><li>Two fructose </li></ul><ul><li>Glucose and galactose </li></ul><ul><li>Glucose and fructose </li></ul><ul><li>Glucose and glucose </li></ul><ul><li>What kind of sugars are found in the disaccharide sucrose? </li></ul><ul><li>Fructose and galactose </li></ul><ul><li>Two glucose </li></ul><ul><li>Glucose and fructose </li></ul><ul><li>two fructose </li></ul>
  5. 5. Polysaccharides <ul><li>Polysaccharides are polymers of monosaccharides also called glycans. </li></ul><ul><li>Polysaccharides differ from each other </li></ul><ul><li>-identity of the recurring monosaccharide units. </li></ul><ul><li>-length of their chains. </li></ul><ul><li>-degree of branching. </li></ul>
  6. 7. <ul><li>Starch in plants and glycogen in animals are the most important storage polysaccharide. </li></ul><ul><li>Both occur as intracellular granules or large clusters. </li></ul><ul><li>Heavily hydrated since they have many exposed hydroxyl groups. </li></ul>Homopolysaccharides as stored fuel
  7. 8. Starch and Glycogen
  8. 9. Starch <ul><li>Contains amylose and amylopectin which are glucose polymers. </li></ul><ul><li>Amylose has long chains of D-glucose. residues connected by ( α1 4) linkage. </li></ul><ul><li>Amylopectin is branched ( α1 6). Branching occur every 24-30 residues. </li></ul><ul><li>Most abundantly found in tubers in plants. </li></ul>
  9. 10. <ul><li>Glycogen is more extensively branched (branch on average at every 8-12 residues). More compact than starch. </li></ul><ul><li>Liver (hepatocytes), skeletal muscles. </li></ul><ul><li>Dextran: used in Size exclusion chromatography. </li></ul>Glycogen
  10. 11. Homoploysaccharides as structural biomolecules <ul><li>Cellulose </li></ul><ul><li>Cellulose is fibrous, tough, and water insoluble substance found in cell walls of plant cells. </li></ul><ul><li>It is linear, unbranched, consisting of D-glucose monomers with ( β 1 4)-linkage. </li></ul>
  11. 12. Cellulose <ul><li>Termites, bacteria, ruminants, cattles, and wood fungus can break down </li></ul><ul><li>cellulose. </li></ul><ul><li>Why animals cannot use cellulose as a fuel? </li></ul>
  12. 13. Chitin <ul><li>Composed of N -acetylglucosamine residues in ( β 1 4)-linkage. </li></ul>
  13. 14. Heteropolysaccharides as structural components <ul><li>Glycosaminoglycans </li></ul><ul><li>repeating disaccharide units </li></ul><ul><li>-either N-acetylglucosamnie or N- acetylgalactosamine. </li></ul><ul><li>-D-glucuronic acid or L-iduronic acid. </li></ul><ul><li>esterified sulfate groups. </li></ul>
  14. 15. <ul><li>Present in ECM. </li></ul><ul><li>Hyaluronan </li></ul><ul><li>Chondritin sulfate </li></ul><ul><li>Keratan sulfate </li></ul><ul><li>Heparan sulfate </li></ul>
  15. 16. <ul><li>Peptidoglycan </li></ul><ul><li>Alternating </li></ul><ul><li>( β 1 4)-linked </li></ul><ul><li>N -acetylglucosamine and </li></ul><ul><li>N -acetylmuramic acid units. </li></ul>
  16. 17. Polysaccharides as information carriers <ul><li>Communication between cell and extracellular components. </li></ul><ul><li>Transportation of proteins. </li></ul><ul><li>Recognition sites for extracellular signal molecules. </li></ul><ul><li>Glycocalyx serves is made up of oligosaccharides. </li></ul>
  17. 18. Glycoconjugates <ul><li>Proteoglycans </li></ul><ul><li>Glycoproteins </li></ul><ul><li>Glycolipids </li></ul>
  18. 19. Glycoproteins <ul><li>Carbohydrate protein conjugates. </li></ul>
  19. 20. Glycoproteins <ul><li>Eg. Mucins, glycophorin A, immunoglobins, and certain harmones, collagen. </li></ul>Oligosaccharide chains are attached either posttranslation or cotranslation modification.
  20. 21. <ul><li>Glycomics </li></ul><ul><li>systematic characterization of all the carbohydrate component of the cell or tissue, including those attached to proteins and to lipids. </li></ul>
  21. 22. <ul><li>What is the biological advantage of adding oligosaccharides to proteins? </li></ul><ul><li>How Lysozyme kills bacteria? </li></ul><ul><li>What is mode of action of penicillin? </li></ul>
  22. 23. Separation techniques for Carbohydrates
  23. 24. Chromatography <ul><li>Mobile phase </li></ul><ul><ul><li>Mixture dissolved in liquid or solid </li></ul></ul><ul><li>Stationary phase </li></ul><ul><ul><li>Porous solid matrix </li></ul></ul><ul><li>Components of mixture pass through the column at different rates based on properties </li></ul>
  24. 25. Gel Filteration <ul><li>Gel-filtration </li></ul><ul><ul><li>Size/molecular exclusion chromatography </li></ul></ul><ul><ul><li>Stationary phase = gels with pores of particular size </li></ul></ul><ul><ul><li>Molecules separate based on size </li></ul></ul><ul><ul><ul><li>Small molecules caught in pores </li></ul></ul></ul><ul><ul><ul><li>Large molecules pass through </li></ul></ul></ul>
  25. 26. Affinity chromatography <ul><li>Affinity </li></ul><ul><ul><li>Matrix chemically altered to include a molecule designed to bind a particular carbohydrate </li></ul></ul><ul><ul><li>Other carbohydrates pass through </li></ul></ul>
  26. 27. Affinity Chromatography <ul><li>Separation is based on highly specific interactions. </li></ul><ul><li>Lectin affinity chromatography is when lectin is used to separate components within a mixture. Lectin binds to carbodydrates. Eg. Concanavalin A </li></ul>
  27. 28. High Performance Liquid Chromatography (HPLC) <ul><li>-Stationary phase = small uniform particles, large surface area </li></ul><ul><li>-a solvent is forced through under high pressures of up to 400 atmospheres. </li></ul><ul><li>-Adapt to separate based on polarity, size, etc. </li></ul>
  28. 29. Mass Spectrometery <ul><li>Determines the mass-to-charge ratio of charged molecules. </li></ul><ul><li>loaded sample is vaporized. </li></ul><ul><li>ionization </li></ul><ul><ul><li>Chemical ionization </li></ul></ul><ul><ul><li>Electrospray ionization </li></ul></ul><ul><ul><li>Matrix assisted laser desorption/ionization (MALDI) </li></ul></ul><ul><ul><li>Ions are separated by electromagnetic field </li></ul></ul>
  29. 30. Oligosaccharides in a group of glycoproteins analyzed by MALDI MS
  30. 31. Learning check! <ul><li>Heparin is routinely added to blood samples obtained for clinical analysis to </li></ul><ul><li>inhibit blood coagulation. </li></ul><ul><li>enhance blood coagulation. </li></ul><ul><li>activate thrombin. </li></ul><ul><li>enhance protease activity. </li></ul><ul><li>Which of the following are components of glycosaminoglycan? </li></ul><ul><li>N-acetylglucosamine </li></ul><ul><li>N-acetlygalactosamine </li></ul><ul><li>Sulfate </li></ul><ul><li>N-acetylmuramic acid </li></ul><ul><li>D-glucuronic acid </li></ul>
  31. 32. <ul><li>Hyaluronidase is an enzyme founf in pathogenic bacteria which hydrolysis </li></ul><ul><li>Chondritin sulfate </li></ul><ul><li>Keratan sulfate </li></ul><ul><li>Heparan sulfate </li></ul><ul><li>hyaluronan </li></ul>
  32. 33. Amino Acids, Peptides, and Proteins
  33. 34. Amino acids <ul><li>The building blocks of proteins </li></ul><ul><ul><li>Essential amino acids </li></ul></ul><ul><li>20 standard amino acids </li></ul><ul><li>Two functional groups: </li></ul><ul><ul><li>carboxylic acid group </li></ul></ul><ul><ul><li>amino group on the alpha (  ) carbon </li></ul></ul><ul><li>Have different side groups (R) </li></ul><ul><ul><li>Properties dictate behavior of AAs </li></ul></ul>
  34. 35. Zwitterions <ul><li>Both the –NH 2 and the –COOH groups in an amino acid undergo ionization in water. </li></ul><ul><li>At physiological pH (7.4), a zwitterion forms </li></ul><ul><ul><li>Both + and – charges </li></ul></ul><ul><ul><li>Overall neutral </li></ul></ul><ul><ul><li>Amphoteric </li></ul></ul><ul><ul><ul><li>Amino group is protonated </li></ul></ul></ul><ul><ul><ul><li>Carboxyl group is deprotonated </li></ul></ul></ul>
  35. 36. Zwitterions <ul><li>Soluble in polar solvents due to ionic character </li></ul><ul><li>Structure of R also influence solubility </li></ul>
  36. 37. Classification of Amino Acids <ul><li>Classify by structure of R </li></ul><ul><ul><li>Nonpolar </li></ul></ul><ul><ul><li>Polar </li></ul></ul><ul><ul><li>Aromatic </li></ul></ul><ul><ul><li>Acidic </li></ul></ul><ul><ul><li>Basic </li></ul></ul>
  37. 38. Non-polar, aliphatic R groups
  38. 39. Polar, uncharged R group
  39. 40. Disulfide Bonds
  40. 41. Aromatic R groups
  41. 42. UV-Vis Spectroscopy <ul><li>Absorbance used to monitor protein concentrations </li></ul>
  42. 43. Acidic and Basic Amino Acids <ul><li>Acidic </li></ul><ul><ul><li>R group = carboxylic acid </li></ul></ul><ul><ul><li>Negatively charged </li></ul></ul><ul><li>Basic </li></ul><ul><ul><li>R group = amine </li></ul></ul><ul><ul><li>Positively charged </li></ul></ul><ul><ul><li>His ionizes at pH 6.0 </li></ul></ul>
  43. 44. Uncommon amino acids <ul><li>4-hydroxyproline </li></ul><ul><li>5-hydroxylysine </li></ul><ul><li>6-N-methyllysine </li></ul><ul><li>Selenocysteine </li></ul><ul><li>Ornithine </li></ul><ul><li>Citrulline </li></ul>
  44. 45. Acid-base Properties <ul><li>AAs also have multiple pK a ’s due to multiple ionizable groups </li></ul>pK 1 ~ 2.2 (protonated below 2.2) pK 2 ~ 9.4 (NH 3 + below 9.4) pK R (when applicable)
  45. 46. 3-letter and 1-letter abbreviations
  46. 47. pH and Ionization
  47. 48. Titration of Glycine <ul><li>pK 1 </li></ul><ul><ul><li>[cation] = [zwitterion] </li></ul></ul><ul><li>pK 2 </li></ul><ul><ul><li>[zwitterion] = [anion] </li></ul></ul><ul><li>First equivalence point </li></ul><ul><ul><li>Zwitterion </li></ul></ul><ul><ul><li>Molecule has no net charge </li></ul></ul><ul><ul><li>pH = pI (Isoelectric point) </li></ul></ul><ul><ul><li>pI = average of pK a ’s = ½ (pK 1 + pK 2 ) </li></ul></ul><ul><ul><li>pI glycine = ½ (2.34 + 9.60) = 5.97 </li></ul></ul>
  48. 49. Titration curve of histidine -Aas with ionizable R groups have three pKa values
  49. 50. pI <ul><li>For AAs with 3 pK a ’s, pI = average of two relevant pK a values </li></ul><ul><li>Consider lysine (pK 1 = 2.18, pK 2 = 8.95, pK R = 10.53): </li></ul><ul><li>Which species is the isoelectric form? </li></ul><ul><li>So, pI = ½ (pK 2 + pK R ) </li></ul><ul><li> = ½ (8.95 + 10.53) = 9.74 </li></ul>
  50. 51. Stereochemistry of AAs <ul><li>All amino acids (except glycine) are optically active </li></ul><ul><li>Fischer projections: </li></ul>
  51. 52. D and L Configurations <ul><li>d = dextrorotatory </li></ul><ul><li>l = levorotatory </li></ul><ul><li>D, L = relative to glyceraldehyde </li></ul>
  52. 53. Importance of Stereochemistry <ul><li>All AA ’s found in proteins are L geometry </li></ul><ul><ul><li>S enantiomer for all except cysteine </li></ul></ul><ul><li>D-AA ’s are found in bacteria </li></ul><ul><li>Geometry of proteins affects reactivity (e.g binding of substrates in enzymes) </li></ul><ul><ul><li>Thalidomide </li></ul></ul>
  53. 54. Learning Check! <ul><li>Glycine side chain is –CH3 </li></ul><ul><li>Alanine side chain is CH2-phenyl </li></ul><ul><li>Phenylalanine side chain is -H </li></ul><ul><li>Cysteine side chain is –CH2-SH </li></ul><ul><li>Proline is the only one of the 20 standard amino acids that lacks a primary amine. </li></ul><ul><li>The side chain of histidine has pK close to neutrality. (T/F) </li></ul>
  54. 55. <ul><li>Give the single letter notation for any one of the polar neutral amino acids. </li></ul><ul><li>S, T, C, N, Q </li></ul>
  55. 56. The Peptide Bond
  56. 57. Polypeptides <ul><li>Linear polymers (no branches) </li></ul><ul><li>Terminal residues: </li></ul><ul><ul><li>Free amino group (N-terminus) </li></ul></ul><ul><ul><ul><li>Draw on left </li></ul></ul></ul><ul><ul><li>Free carboxylate group (C-terminus) </li></ul></ul><ul><ul><ul><li>Draw on right </li></ul></ul></ul><ul><li>pK a values of AAs in polypeptides differ slightly from pK a values of free AAs </li></ul>
  57. 58. Naming Peptides <ul><li>Name from the free amine (NH 3 + ) </li></ul><ul><li>Use - yl endings for the names of the amino acids </li></ul><ul><li>The last amino acid with the free carboxyl group (COO - ) uses its amino acid name </li></ul>
  58. 59. Learning Check! Ser-Gly-Tyr-Ala-Leu serylglycyltyrosylalanylleucine
  59. 60. Calculate number of AAs present in a protein <ul><li>For a simple protein </li></ul><ul><li>Molecular weight / 110 </li></ul>
  60. 61. Separation and purification <ul><li>Determine a source (tissue) </li></ul><ul><li>Extract protein </li></ul><ul><ul><li>Suspend cell source in buffer </li></ul></ul><ul><ul><li>Homogenize </li></ul></ul><ul><ul><ul><li>Break into fine pieces </li></ul></ul></ul><ul><ul><ul><li>Cells disrupted </li></ul></ul></ul><ul><ul><ul><li>Soluble contents mix with buffer </li></ul></ul></ul><ul><ul><ul><li>Centrifuge to separate soluble and insoluble </li></ul></ul></ul><ul><li>Separate protein of interest </li></ul><ul><ul><li>Based on solubility, size, charge, or binding ability </li></ul></ul>
  61. 62. Concentration of salts <ul><li>Salting out </li></ul><ul><ul><li>Continue to increase [salt] decreases [protein] </li></ul></ul><ul><li>Different proteins salt out at different [salt] </li></ul><ul><li>Ammonium sulfate is commonly used salt. </li></ul>
  62. 63. Types of Chromatography <ul><li>Paper </li></ul><ul><ul><li>Stationary phase = filter paper </li></ul></ul><ul><ul><li>Same theory as thin layer chromatography (TLC) </li></ul></ul><ul><ul><li>Components separate based on polarity </li></ul></ul><ul><li>High-performance liquid (HPLC) </li></ul><ul><ul><li>Stationary phase = small uniform particles, large surface area </li></ul></ul><ul><ul><li>Adapt to separate based on polarity, size, etc. </li></ul></ul>
  63. 64. Types of Chromatography <ul><li>Ion-exchange chromatography </li></ul><ul><li>Gel filtration / size-exclusion chromatography </li></ul><ul><li>Affinity chromatography </li></ul>
  64. 65. Electrophoresis Separation of proteins based on their charge in an electric field.
  65. 66. <ul><li>-binds to proteins and gives them </li></ul><ul><li>a similar charge-to-mass ratio. </li></ul><ul><li>-Unfolds the proteins so that </li></ul><ul><li>all assume similar shape. </li></ul>
  66. 67. Isoelectric focusing <ul><li>-determining isoelectric point of a protein. </li></ul><ul><li>-Gradient gel is prepared by ampholytes. </li></ul>
  67. 68. <ul><li>-2D electrophoresis </li></ul><ul><li>-separation of proteins </li></ul><ul><li>With identical molecular </li></ul><ul><li>Weight but different pI </li></ul>
  68. 69. <ul><li>E-mail address of one student required. </li></ul><ul><li>Electronic submission of assignments. </li></ul>
  69. 70. Agar <ul><li>Present in cell walls of marine algae and sea weeds. </li></ul><ul><li>D-galactose and an L-galactose derivative ether linked between C3 and C6 </li></ul><ul><li>Agarose solid support to grow bacterial colonies. Capsules in which some vitamins and drugs are packaged. </li></ul>
  70. 73. Ion Exchange Chromatography <ul><li>Ion-exchange </li></ul><ul><ul><li>Stationary phase = chemically modified to include charged groups </li></ul></ul><ul><ul><li>Separate based on net charge of carbohydrates </li></ul></ul><ul><ul><li>Anion exchangers </li></ul></ul><ul><ul><ul><li>Cation groups (protonated amines) bind anions </li></ul></ul></ul><ul><ul><li>Cation exchangers </li></ul></ul><ul><ul><ul><li>Anion groups (carboxylates) bind cations </li></ul></ul></ul>
  71. 74. Ion Exchange Chromatography
  72. 75. Affinity Chromatography
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