Hi, everyone,
I am trying to express 14 kD protein in E.coli BL21 DE3. Then i purified this protein using DEAE-sepharose column by eluting with NaCl. When I checked different fractions on SDS-PAGE, single band was observed. After desalting, lyophilization, and redissolved in tris buffer, it initially show single band but after putting for some days at 4 degree, low molecular bands are observed. Sometime it was degraded completely even at 4 degree.
What could be the reason?
I am using 1 mM PMSF for the cell lysis.
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#2
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an elder
Posted 25 September 2009 - 07:03 AM
you need more than just pmsf to inhibit the range of proteases present. most labs use a inhibitor cocktail.
also, pmsf is quite labile. it an decompose in as little as 30 minutes under some fairly normal storage conditions so will not offer protection against additional proteases (contributed by things growing in your sample).
also, pmsf is quite labile. it an decompose in as little as 30 minutes under some fairly normal storage conditions so will not offer protection against additional proteases (contributed by things growing in your sample).
talent does what it can
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genius does what it must
i do what i get paid to do
#3
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member
Posted 29 September 2009 - 06:11 AM
Thanks mdfenko for suggestion. I will try this.
