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Descrição

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Maryam Ghanem
Fluxograma por Maryam Ghanem, atualizado more than 1 year ago
Maryam Ghanem
Criado por Maryam Ghanem aproximadamente 7 anos atrás
6
0

Resumo de Recurso

Nós do fluxograma

  • Separation of serum protein on SDS-PAGE
  • Set up the apparatus as in figure 8 to pour a gel , using the casting stand and spacers of 1.5 mm
  • Prepare the running gel in 100 ml beaker 
  • Pour the freshly mixed running gel in to space between two parallel glass plates in the casting stand 
  • Add water layer above the running gel , allow the gel to polymerize for at least 30 min , then pour of the water from the polymerized running gel 
  • Prepare the stacking gel 
  • Pour the stacking gel in to the running gel 
  • 17.8 ml distilled water 
  • 13.6 ml 30% acrylamide solution 
  • 8 ml 1.875 M Tri-HCl , pH 8.8 
  • 400 Ml 10% SDS 
  • 20 Ml TEMED 
  • 200 Ml freshly dissolved ammonium persulfate 
  • 3.75 ml distilled water 
  • 0.68 ml 30% acrylamide solution 
  • 0.5 ml 0.6 M Tris-HCl ,pH 8.8 
  • 50       10% SDS 
  • 5      TEMED
  • 25     freshly dissolved ammonium persulfate 
  • Insert the comb to create sample wells in the stacking gel , allow the gel to polymerize to 30 min at RT 
  • Place thetus  polymerized gel in to  the plates into the electrophoresis appara
  • Add running buffer to the chambers at the bottom and top of the gel , remove the comb from between the plates 
  • Prepare the standard  and unknown protein samples 
  • mix 10 Ml of each with 30 Ml of sample buffer , mix and heat at 100 C for 5 min 
  • Load the samples onto the gel with a syringe 
  • Connect the electrode of the apparatus to the power source 
  • Apply a 50 mamp current , 200 V  to the apparatus and continue electrophoresis until the bromophenol blue reach the bottom of the gel 
  • Turn off the power and disconnect the electrodes , remove the gel from apparatus , by sepatula , separat the two plates and separate the soft stacking gel from the more rigid running gel 
  • Transfer the running gel into pan filled with 50 ml of coomassie blue solution allow it to soak for 30 min  
  • Pour off the staining solution and replace it with destaining solution for one hour , replacing it with fresh destainung solution every 15 min 
  • Destaining is complete when the protein appaer as blue bands on transparent gel 

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