1234 1810M1wt1-165 purification
Protein Purification
[edit]2014/12/12-2014/12/19
SHANYI
1234 pET30b-M1wt-1-165
culrured on 12/13/2014
Tuner(DE3)plysN ( transformed on Dec.12), culture overnight 0.2mM IPTG RT induce 2hour, cell lysed
lysis buffer PH is 8.0, containing 10mM Imidazole
lane5-12: 1234 M1wt1-165 eluted from Ni column (elution buffer:25 mM NaPO4 pH 4.0, 250 mM Imidozole, 200 mM NaCl,)
combine,dilute with 4cv dilution buffer(25 mM NaPO4 pH 5.0), load to 25 ml spff
connect to FPLC, wash column with 20mM Tris-Hcl PH8.0 50mM NaCL, then elute with 50-500ml Nacl PH8.0, finally use 1M Nacl to elute protein
lane1: 1234 protein eluted from SPFF ( method run 12/16/2014) fraction 25 ==>250mM Nacl
lane2: 1234 protein eluted from SPFF ( method run 12/16/2014) fraction 46==>450mM Nacl
lane3: 1234 protein eluted from SPFF ( method run 12/16/2014) fraction 49==>1M Nacl
lane4: 1234 pET30b-M1wt-1-165, culrured on 12/15/2014, lysate supernatant(Tuner(DE3)plysN , 0.4mM IPTG RT induce O/N, 15.5g/4L)
lane5: 1810 pT7A30-M1wt-1-165, culrured on 12/15/2014, lysate supernatant(Tuner(DE3)plysN , 0.2mM IPTG RT induce O/N, 15.5g/4L)
lane6: marker
load 1234,1810 lysate to Ni column,
wash with bufferA, then wash with 35mM Imidazole, elute with elution buffer(25 mM NaPO4 pH 4.0, 250 mM Imidozole, 200 mM NaCl,)
lane6: Ni column 10mM Imidazole wash flowthrough
lane7-8:Ni column 35mM Imidazole wash flowthrough
lane9-12: protein eluted from Ni column
combine,dilute with 4cv dilution buffer(25 mM NaPO4 pH 5.0), load to 25 ml spff
connect to FPLC, wash column with 20mM NaPO4 PH7.0, 50mM Nacl, then elute with 50-500ml Nacl PH8.0, finally use 1M Nacl to elute protein
lane1-3: protein eluted from spff( 12/16/2014, f43,f45,f48)
lane4-8: protein eluted from spff(12/18/2014,f9,f11,f29,f31,f44)
dissolve pellet in 4M urea PH8.0 buffer,load to Ni column
lane10: dissolved protein
lane11: Ni flowthrough