Purification of protein 9

Running Head: PURIFICATION OF PROTEIN
Purification of Protein
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Purification of Protein
1
Purification of protein 9
The protein 9 has an enzyme activity that is stable for several hours at a temperature of 50
degrees centigrade and a pH of 4.0 to 9.5.
The protein of interest from the immunoblot image shows that it has an isoelectric pH of 6.8
and molecular weight approximately 25kDa.
The initial default enzyme mixture was subjected to heat denaturation for 20 minutes at 50
degrees centigrade.
Then further junk undesired proteins were removed with the help of 70% Ammonium
sulphate fractionation.
The hydrophobic interaction purification method was exploited to remove the proteins based
on a salt gradient of 0 to 1.0 molar. This resulted in a loss of 4% of the protein in the initial
buffer but the enzyme activity was not hampered.
The protein mixture is subjected to gel filtration exploiting Bio-gel 60 resin. This was done to
remove all the low molecular weight proteins from the default mixture. The desired protein
was eluted at the end of the elution peak and fractions were collected accordingly.
Maintaining a pH gradient 6.0 to 7.0 with pH of the buffer at 6.5 ion exchange
chromatography was exploited using Q-sepharose resin.
The gel image of the purified protein is shown below: