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Journal of Mosquito Research, 2013, Vol.3, No.4, 21
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32
ISSN 1927-646X
http://jmr.sophiapublisher.com
24
300 mM NaCl and 10 mM imidazole, pH 8.0) at ratio
of 2 to 5 mL per gram wet weight. Lysozyme was
added to a final concentration of 1 mg/mL and
incubated on ice for 30 min. The protease inhibitor,
phenylmethylsulfonyl fluoride (PMSF), was then
added to a final concentration of 3 mM to prevent
protein denaturation. Total soluble proteins were
extracted by sonication in ice bucket (4 times, 30 s
each). The cell debris was removed by centrifugation
at 5 000 rpm for 15 min at 4
℃
; the supernatant
containing the soluble Tsf was used directly or stored
at −20 until needed. The purification of the
℃
recombinant Tsf protein from total soluble bacterial
extract was performed using Ni-NTA agarose resin
(QIAGEN) according to the manufacturer’s
instructions. The amount of cells required depends on
the expression level of the 6× His- Tagged protein and
the expression system used. After batching the
bacterial extract with Ni-NTA resin, the resin was
washed with wash buffer (50 mM NaH
2
PO
4
; 300 mM
NaCl and 20 mM immidazole, pH8.0) and Tsf was
then eluted with elution buffer (50 mM NaH
2
PO
4
, 300
mM NaCl and 250 mM immidazole, pH 8.0) in a
minimal volu