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CH339K

CH339K. Physical Methods: How to Purify and Sequence a Weapons-Grade Protein. First Question. How do I measure the amount of protein I have?. UV Absorption Spectrophotometry. Second Question. How can I spot my protein in the great mass of different proteins?. Electrophoresis.

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CH339K

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  1. CH339K Physical Methods: How to Purify and Sequence a Weapons-Grade Protein

  2. First Question How do I measure the amount of protein I have?

  3. UV Absorption Spectrophotometry

  4. Second Question How can I spot my protein in the great mass of different proteins?

  5. Electrophoresis

  6. The frictional coefficient f depends on the size of the molecule, which in turn depends upon the molecular mass, so: i.e. the velocity depends on the charge/mass ratio, which varies from protein to protein

  7. Polyacrylamide Gels

  8. Polyacrylamide gel electrophoresis of whole cell proteins of three strains of lactic acid bacteria.

  9. Agarose Gelidium sp.

  10. SDS PAGE Sodium Dodecyl (Lauryl) Sulfate SDS binds to proteins at a constant ratio of 1.4 g SDS/g protein

  11. Constant q/M ratio

  12. Disulfide cleavage

  13. Disulfide cleavage and chain separation + bME

  14. Isoelectric Point

  15. Isoelectric Focusing

  16. pH

  17. Carrier Ampholytes • Amphoteric Electrolytes • Mixture of molecules containing multiple amino- and carboxyl- groups with closely spaced pIs • Partition into a smooth, buffered pH gradient

  18. Separation by pI

  19. Isoelectric Focusing Below the pI, a protein has a positive charge and migrates toward the cathode Above the pI, a protein has a negative charge and migrates toward the anode

  20. Isoelectric FocusingFoot Flesh Extracts from Pomacea flagellata and Pomacea patula catemacensis

  21. Protein Purification Steps 1 unit = amount of enzyme that catalyzes conversion of 1 mmol of substrate to product in 1 minute

  22. Purification visualized

  23. Example:Purification of Ricin

  24. Georgi Markov 1929-1978

  25. Ricinus communis – castor oil plant

  26. Ricin Ricin B chain (the attachment bit)

  27. Ricin Action • Ricin and related enzymes remove an adenine base from the large ribosomal RNA • Shut down protein synthesis

  28. The possibility that ricin might be used as an asymmetric warfare weapon has not escaped the attention of the armed services. The last time I was qualified to know for sure, there were no effective antidotes.

  29. Raw Extract (NH4)2SO4 Cut Affinity Gel Filtration

  30. Salting In – Salting out • salting in: Increasing ionic strength increases protein solubility • salting out: Increasing further leads to a loss of solubility

  31. Salting in – salting out The solubility of haemoglobin in different electrolytes as a function of ionic strength. Derived from original data by Green, A.A. J. Biol. Chem. 1932, 95, 47

  32. Salting in: Counterions help prevent formation of interchain salt links Solubility reaches minimum at pI

  33. Salting out: there’s simply less water available to solubilize the protein.

  34. Different proteins have different solubilities in (NH4)2SO4

  35. Lyotropic  ChaotropicSeries Cations: N(CH3)3H+> NH4+> K+> Na+> Li+> Mg2+>Ca2+>Al3+> guanidinium / urea Anions: SO42−> HPO42−> CH3COO−> citrate > tartrate > F−> Cl−> Br−> I−> NO3−> ClO4−> SCN−

  36. Bring to 37% Saturation – ricin still soluble, many other proteins ppt • Collect supernatant • Bring to 67% Saturation – ricin ppt, many remaining proteins still soluble • Collect pellet • Redissolve in buffer

  37. Dialysis and Ultrafiltration(How do you get the %@$&#! salt out?)

  38. Raw Extract (NH4)2SO4 Cut Affinity Gel Filtration

  39. Separation by chromatography Basic Idea: You have a stationary phase You have a mobile phase Your material partitions out between the phases.

  40. Affinity Chromatography

  41. Structure of Agarose Agarose is a polymer of agarobiose, which in turn consists of one unit each of galactose and 3,6-anhydro-a-L-galactose. Ricin sticks to galactose, so store-bought agarose acts as an affinity column right out of the bottle, with ricin binding the beads while other proteins wash through.

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