Lab Practical 1

• What is SDS-PAGE?


  Sodium dodecyl sulphate polyacrylamide gel electrophoresis.
• What is Pyruvate Kinase?

  Enzyme that catalyses final step in glycolysis pathway:$phosphoenolpyruvate +$$ADP -> pyruvate +$$ATP$
• Why are RBC reliant on glycolysis to make ATP?

  They don't have mitochondria.
• Symptoms of PK deficiency:

  Lethargy & varying degrees of anaemia ( constant or episodic).
• What is the experiment task?

  Analyse purified PK from blood samples
• What volume does a P20 pipette carry?

  2 - 20 microlitres
• What volume does a P200 pipette carry?

  20 - 200 microlitres
• What volume does a P1000 pipette carry?

  200 - 1000 microlitres
• What volume does a P5000 pipette carry?

  1000 - 5000 microlitres
• What does SDS-PAGE do?


  Separate proteins on basis of their molecular weight.
• What is SDS?


  Anionic detergent that denatures proteins, removing all native structure & coats them in negative charge due to the sulphate groups.
• What is DTT?


  Dithiothreitol- strong reducing agent that breaks any disulphide bonds between cysteine residues in the proteins.
• What is the gel made of?

  Polyacrylamide
• What is applied to the gel after samples have been loaded?

  Electrical voltage
• Where do the denatured proteins move?

  Towards positive pole due to negative charge added by SDS. Smaller proteins move more quickly through the gel than large proteins as they can more easily fit through the pores in polyacrylamide.
• What are the samples first mixed with?

  A sample loading buffer containing SDS, DTT & a blue dye (bromophenol blue). Also contains high concentration of glycerol which makes buffer dense so samples sink to bottom of well when applied to gel.
• What are the samples heated to before loading?

  95 degrees Celsius for 5 mins to ensure complete denaturation by SDS & DTT.
• What happens to the gel after electrophoresis?

  Stained using blue dye (Coomassie) which binds to the proteins.
• What are marker proteins?

  Have known molecular weights- loaded onto 1 well of the gel. Used to estimate molecular weight of other proteins. Marker proteins pre-stained so can be seen as gel runs.