Editing Purification of His-GST Fusion Protein (section)

= Expression and Purification of His-GST-Tagged Protein in pCold Vector&nbsp; =

== <br/>1. Protein Expression&nbsp; with Nico21(DE3)pLysN. ==

Inoculate of&nbsp; 500 ml&nbsp; LB&nbsp;K/C&nbsp;in a 500&nbsp;ml&nbsp; flask&nbsp; with&nbsp;Nico21(DE3)pLysN ( from glycerol stock transformed with desired plasmid&nbsp;)

Shake overnight at 37˚C

Next Morning, aliquot&nbsp; culture to 8 flasks of&nbsp; 1 liter&nbsp;LB&nbsp;K/C&nbsp;

Grow to OD600 =&nbsp;1.0 to 1.2 at 37 oC shaker

Take out&nbsp; flasks, cool to room termperature.

Add 0.5 ml 1 M IPTG to each flask.

Take flasks to an incubator in the cold room.

Induce O/N (12~24&nbsp;hours at 15 oC.&nbsp;

Spin down cells, combine into one 500 ml centrifuge tube

Freeze o/n.

== 2. Purification On Ni column ==

Add 450 ml cold Buffer A to frozen cells

Lyse and homogenize

Spin 25000 rpm for 30 min at 4 oC, using ultracentrifuge

Combine supernatant.

Check pellet to see if it is loose or sticky

Load supernatant to regenerated Ni beads (10 ml)

Wash with 500 ml Buffer A.

Elute protein with 100 ml Buffer B.

Collect 10 ml fractions using a fraction collector

Measure protein concentration in each fraction.

== 3. Purification using Glutathione Beads ==

Pour fractions with protein to a column packed with 2 ml GE&nbsp;Glutathione Beads.

Wash column with 20 ml PBS

Elute His-GST fusion protein with 10 X 1ml of '''Glutathione Elution Buffer'''

Collect 1 ml fractions

Check protein concentration in each fraction

Run SDS-Gel to check purity.

Combine fractions containing desired protein.

Optional: Concentrate protein using 9K spin concentor.

Concentrate protein by dialysis vs Storage Buffer (containing 50% glycerol). Add 1 M DTT to final concentration of 5 mM.

Record protein purification result on database

Print labels, label protein vials and store at -80&nbsp;oC.

== <br/>Buffers ==

[[Ni_Buffer_A|'''Buffer A''']]

[[Ni_Buffer_B|'''Buffer B''']]

'''Glutathione Elution Buffer'''<br/>100&nbsp;mg Reduced Glutathione (Sigma) in 10 ml PBS<br/><br/>
[[Category:Protocols]]