Materials

• StockAcrylamide:(30%T:0.8%C)
  • 30%byweightofacrylamide
  • 0.8%byweightofN,N"-bis-methyleneacrylamide
• SeparationGel(FinalConcentrations)
  • 10%acrylamide(1:3dilutionofstock)
  • 0.375MTris-HCl(pH8.8)
  • 0.1%SDS
• StackingGel(FinalConcentrations)
  • 3%acrylamide(1:10dilutionofstock)
  • 0.125MTris-HCl(pH6.8)
  • 0.1%SDS
• ElectrodeBuffer
  • 0.025MTris
  • 0.192MGlycine
  • 0.1%SDS
  • AdjustpHto8.3
• 0.2-0.3mlsamplesofbrainextract,andcontaining
  • 200microgramsprotein
  • 0.0625MTris-HCl(pH6.8)
  • 2%SDS
  • 10%glycerol
  • 5%-mercaptoethanol
  • 0.001%bromophenolblue
• TEMED
• 10%(w/v)Ammoniumpersulphate
• 50%and7%(w/v)TCA(trichloroaceticacid)
• 0.1%(w/v)Coomassiebrilliantbluein50%TCA

Procedure

1. Prepare15cmglasstubeswith6mminternaldiameter.Polymerizea10cmseparationgelanda1cmstackinggelwithinthetubesbytheadditionofTEMEDandammoniumpersulfateasdirectedinChapterFour.

2. Assemblealltheequipmentandplace200µlofsampleinonetube.Setupasecondgelcontaining200µlofproteinmolecularweightstandards.

3. Electrophoresisiscarriedoutat3mApergeluntilthebromophenolbluedyereachesthebottomofthetube(approximately7hours).

4. Fixthegelsovernightin50%TCA.

5. Stainwith0.1%Coomassiebrilliantblue(freshin50%TCA)for1hourat37°C.

6. Diffusion-destainwithrepeatedwashingin7%TCA.

7. Scanthegelsorphotographicallyanalyzethem.

8. Determinethemolecularweightsoftheproteins(tubulinandMAP"s).

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