I
Induction (Small Scale)
-Grow a 5 ml o/n culture.
-Inoculate 2 ml fresh media with 200 µl of
o/n culture & grow for 2 h (@ 37°C with
shaking).
-Induce for 4 h (add 1 µl of 500 mM IPTG.
-Spin down 1.5 ml.
-Resuspend in 300 µl PBS.
-Add 100 µl 4X Reducing Buffer.
-Boil for 10 min.
-Subject to SDS-PAGE (Tricine Gels).
-Grow a 5 ml o/n culture.
-Inoculate 2 ml fresh media with 200 µl of
o/n culture & grow for 2 h (@ 37°C with
shaking).
-Induce for 4 h (add 1 µl of 500 mM IPTG.
-Spin down 1.5 ml.
-Resuspend in 300 µl PBS.
-Add 100 µl 4X Reducing Buffer.
-Boil for 10 min.
-Subject to SDS-PAGE (Tricine Gels).
Induction (Large Scale)
-Autoclave 1 L LB/protein to be expressed.
-Grow 25 ml o/n cultures of respective clones to
express (using LB-Amp).
-Ampicillin added at 1 µll/ml of media (100 µlg/ml
stock).
-Add the 25 ml o/n culture + 1 ml Amp to the 1 L
flask of LB.
-Grow for 2 h with shaking (200 rpm) at 37°C.
-Take a 1 ml aliquote and check O.D.600 and
save as the preinduction sample (store @ 4°C).
O.D. should be around 0.6-0.8.
-Induce with 0.2 mM IPTG (400 ul of a 500 mM
stock).
-Grow for an additional 4 h and take aliquote for
an O.D.600 reading (Store @ 4°C).
-Spin I L LB culture @ 5000 RPM/15 min.
-Pour supernatant back into flask and autoclave
(waste).
-Resuspend pelet in 1X PBS; put into a 50 ml
tube, four off suppernatant and add 25 ml
Binding Buffer.
-Freeze at -20°C if to be cracked the next day or
at -70°C for longer storage.
-Crack using the French Press.
-Purify proteins using a Ni-column.
-Autoclave 1 L LB/protein to be expressed.
-Grow 25 ml o/n cultures of respective clones to
express (using LB-Amp).
-Ampicillin added at 1 µll/ml of media (100 µlg/ml
stock).
-Add the 25 ml o/n culture + 1 ml Amp to the 1 L
flask of LB.
-Grow for 2 h with shaking (200 rpm) at 37°C.
-Take a 1 ml aliquote and check O.D.600 and
save as the preinduction sample (store @ 4°C).
O.D. should be around 0.6-0.8.
-Induce with 0.2 mM IPTG (400 ul of a 500 mM
stock).
-Grow for an additional 4 h and take aliquote for
an O.D.600 reading (Store @ 4°C).
-Spin I L LB culture @ 5000 RPM/15 min.
-Pour supernatant back into flask and autoclave
(waste).
-Resuspend pelet in 1X PBS; put into a 50 ml
tube, four off suppernatant and add 25 ml
Binding Buffer.
-Freeze at -20°C if to be cracked the next day or
at -70°C for longer storage.
-Crack using the French Press.
-Purify proteins using a Ni-column.
IPTG (500 mM)
(isopropyl-beta-D-thiogalactopyranoside)
1 g IPTG + 8.4 ml H2O (500 mM).
Filter through a 0.22 µM filter.
Store @ -20°C.
(isopropyl-beta-D-thiogalactopyranoside)
1 g IPTG + 8.4 ml H2O (500 mM).
Filter through a 0.22 µM filter.
Store @ -20°C.
IDA Column (1 ml Nickel Column)
-Prepare 1 ml column of pre-charged
Ni-Sepharose Fast Flow (Amersham
Biosciences).
-10 vol Binding Buffer (Buffer A: 25 mM
Tris; 100 mM NaCl, pH 7.9).
-Load Protein (10-20 ml).
-10 vol Binding Buffer.
-Add 10 ml of 10% Buffer B diluted in
Buffer A (Buffer B is same as Buffer A
but with 0.5 M Imidazole).
-Follow the 10% wash with 20%, 30%,
40%, 50%, 60% elutions.
-His-tag protein should be here
somewhere!! Run each elution on an
SDS-PAGE gel to determine where the
protein is and its purity.
-Wash column with 10 vol of 100%
Buffer B.
-Wash then with Buffer A to remove
traces of imidazole
-Wash with 10 vol 20% EtOH and store in
20% EtOH.
ALSO SEE PHOSPHATE BUFFERS.
-Prepare 1 ml column of pre-charged
Ni-Sepharose Fast Flow (Amersham
Biosciences).
-10 vol Binding Buffer (Buffer A: 25 mM
Tris; 100 mM NaCl, pH 7.9).
-Load Protein (10-20 ml).
-10 vol Binding Buffer.
-Add 10 ml of 10% Buffer B diluted in
Buffer A (Buffer B is same as Buffer A
but with 0.5 M Imidazole).
-Follow the 10% wash with 20%, 30%,
40%, 50%, 60% elutions.
-His-tag protein should be here
somewhere!! Run each elution on an
SDS-PAGE gel to determine where the
protein is and its purity.
-Wash column with 10 vol of 100%
Buffer B.
-Wash then with Buffer A to remove
traces of imidazole
-Wash with 10 vol 20% EtOH and store in
20% EtOH.
ALSO SEE PHOSPHATE BUFFERS.
