1810 pT7A30-M1wt-1-165

Cloning of 1810 pT7A30-M1wt-1-165

Purpose: Cloning of1810 pT7A30-M1wt-1-165

Date: 07, 30, 2013

Notebook: 

Page:

Clone ID: 1810

Experimenters: Shanyi

Results

Cloning

subclone from subclone from 1234 pET30b-M1wt-1-165 to 1727pT7A30 using Nde1 + Xho1

 Colony screen

digest with Nde1 + Xho1

Conclusion

Purification

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 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

Purification

12/26/2014----12/29/2014

1810 pT7A30-M1wt-1-165, Tuner(DE3)plysN,  lot number: 80-1810 6mg, 3mg/ml

culrured on 12/25/2014 in LB containing NaPO4+Glucose, 30oC grow overnight, OD600 very high, 0.5mM IPTG 30oC induce 5hours, 8L, 60g

add lysis buffer, freeze and thaw once, 25000rpm 25min, wash pellet with lysis buffer twice

load supernant to Ni column, wash with lysis buffer

elute with elution buffer(25 mM NaPO4 pH 4.0, 250 mM Imidozole, 200 mM NaCl,1mM beta-ME)

dilute with 5%glycerol, protein aggregated, add H3PO4 until it became clear

load to 25ml spff

wash with 100ml 25 mM NaPO4 pH 5.0 ,

elute protein with 0-500 mM NaCL in  25 mM NaPO4 pH 5.0 (linear gredient), then elute protein with 1M Nacl in 25 mM NaPO4 pH 5.0

last, use 0-1M Nacl in PH8.0 buffer to elute protin

protein eluted from spff

lane1-9: protein eluted by 1M Nacl in 25 mM NaPO4 pH 5.0 buffer

lane11,12: protein eluted by 0-1M Nacl in 25 mM Tris-Hcl  pH 8.0  buffer