User:Daniel-Mario Larco/Notebook/AU Photosynthesis Lab/2014/07/21: Difference between revisions
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== | ==Purifying hemoglobin== | ||
#Collect frozen protein solution from last week and thaw | |||
#Once thawed, the solution's pH was changed using concentration device for centrifuge | |||
##15mL of protein solution was added to 45mL of pH8.3 50mM Tris buffer | |||
##The solutions were spun at 3000rpm for 30 minutes | |||
#Repeat the last step | |||
##The solutions were spun until the final total volume was less than 40mL | |||
#Spin down concentrated protein solution to remove impurities | |||
#Equilibrate Q-Sepharose column with 50mM Tris buffer pH8.3 | |||
#Inject 10mL sample of protein into column | |||
##Collect elute during injection | |||
#Once loaded, run a gradient from 0 to 160mM NaCl in Tris pH 8.3 over 10 minutes collected in 5mL fractions | |||
##A peak was collected in this range | |||
##tube labelled fractions 11,12,13 | |||
##concentration is raised to 300mM | |||
##Another set of fractions is collected and placed in tube labelled fractions 14-23 | |||
###These fractions were placed in a centrifuge filter with 50mL 10mM pH 7.2 phosphate buffer for 1 one hour at 3500rpm and 4C | |||
###Repeated twice keeping the two sets of fractions separate | |||
###The initial and final elutes from the column (brown) were stored in the fridge along with the rest of the protein that wasn't used | |||
#The SP-Sepharose column was attached and equilibrated with 10mM pH7.2 sodium phosphate buffer | |||
#10mL of each set of fractions were loaded separately | |||
#The run-through was collected in tubes labeled with the corresponding fractions | |||
All solutions stored in fridge overnight | |||
Revision as of 07:39, 22 July 2014
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Purifying hemoglobin
All solutions stored in fridge overnight
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