Purification and characterization of EGFP Purification and characterization of EGFP by recombinant by recombinant Escherichia coliEscherichia coli

BarneyBarney

05/10/1905/10/19

KanR

PT7lac

NdeI BglII XhoI

LacI

PCMV

EGFP

XhoIBglII

KanR

PT7lac

LacI

pEGFP-C1

EGFP

EGFP

pET30b(+) Polymerase chain reaction

ligation

Chemical transformationChemical transformation 1. Make the competent cell 1. Make the competent cell

Cultivateover-night

E. Coli. cultivate in 3 ml LB

Magnification E. Coli. magnified in 30 ml LB

Shake for about 1 hr

Centrifuge for 5 mins, 500rpm

Discard the super

Suspend with CaCl2

Centrifuge for 5 mins, 500rpm

Discard the super

Suspend with CaCl2, then add 50% glycerol

Allot to the micro-tube

200 l

(cont’d) (cont’d) 2. Transfer2. Transfer

KanR

PT7lac

LacI

Mix together and then place in the ice for 30 mins

Treat with 42℃ for 2 mins

Treat with the ice-bath againfor 5 mins

Cultivate with 1 ml LB

Shake for 1 hr

Spread on the culture medium

Cultivate for 12~16 hrs

TOP 10

CultivationCultivation

Pick any 12 dots randomly

Screen

Cultivate for 12~16 hrs

Colony PCR

Extract plasmid DNA

Transform into competent cell of BL21(DE3)

Cultivate for 12~16 hrs

Magnification

E. Coli. magnified in 30 ml LB

Pick up one line to cultivate in 1 ml LB

(cont’d)(cont’d)

To induce EGFP to be made

IPTG (0.1mM)

React with 2~3 hrs

Centrifuge for 5 mins, 500rpm

Break with supersonic

Suspend withPB buffer

Purify with IMAC NEXT PAGE

IMAC (IMAC (Immobilize Metal Affinity Chromotography)Immobilize Metal Affinity Chromotography)

H2O Starting buffer Target protein

Elution buffer

Starting buffer

Purified EGFP

EDTAH2O

recovery

Western-Blot analysisWestern-Blot analysis

milk

1st

2nd

NEXT PAGE

In Caps Buffer

Wash by TBST Buffer

Wash by TBST Buffer

Blocking Buffer

Anti-polyHistidineAnti-mouse IgG alkaline phosphates-conjugate

(cont’d)(cont’d)

2nd

Wash by TBST Buffer

NBT stock solution

BCIP stock solution

in Alkaline assay buffer