Paul.xu at 17:45, 9 November 2012

2012-11-09T17:45:18Z

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10-15-2012 by xiuli

== 1375- pDB.His.MBP-3C, NiCo21(DE3)PlysN, 0.3mM IPTG, 37 oC, 4 hours ==

Cells: NiCo21(DE3) PlysN was transformed with 1375 pDB.His.MBP-3C. Grow on LB/K/ChL plate for o/.

Grow 1 L LB/K/Chl with 5 ml overnight culture from a single colony,  grow at 37 oC to about OD600 = 0.8.

Induce at 37oC with 300 ul of 1M IPTG to 1 L culture for  4 hours. 

Lysis: Freeze and thaw one time, cells were completely lysed after one freeze-thawing cycle, Use homogenizer to break sticky genomic DNA.

Preclear: Centrifuge at 12000 rpm for 15 min, spin supernatant at 25000rpm for 20 mins. Collect supernatant.

 Purification: 

Add 2 ml Ni beads to a column and equilibrate with lysis buffer. Load clarified supernatant on the column. collect the flow through and load again (if necessary, load one more time to allow protein fully binding), wash with washing buffer with 20 mM imidazole until no detectable protein.

Elute (300 mM imidazole), collect 1 ml fraction each, 7 fraction total.  Detect protein with Biorad reagent. Run fraction 2 on the gel ( protein one) .(  One dark band MBP-3C protein but smaller size), also  the protein  is not pure)

Load 2 ml (slurry) Amylose beads on a column and equlibrate with PBS buffer. Load the above combined fraction 2,3 ,4 on the column and washed with PBS.  Elute with 10mM Maltose in PBS, collect 1ml fraction for total 7 fractions. Run gel. (fraction 2)

[[Image:1375 Ni column purification.jpg]] [[Image:102412 1375 amylose .jpg]] 

 

 

The last lane is MBP-3C protein, between 45kD and 66kD. The size looks samller for fusion protein, maybe only expressed MBP. 

 

Storage: Fraction 2 (OD=2.846) and Fraction 3 (OD= 1.725) was stored at 4 oC in Xiuli's box. Labeled as: 1375 pDB.MBP.3C protein. 

 

[[Category:Protein_Purification]]

PDB.His.MBP-3C - Revision history

Paul.xu at 17:45, 9 November 2012