Editing T4 DNA Ligase 20121223 (section)

== Method Development<br>  ==

Date: Dec, 2012 

Protein eluted from Ni is very dirty: 

[[Image:T4 DNA ligase purification Ni column Lot 20121223.jpg]] 

Dilute protein from Ni column eluate to 50 mM NaCl. Load to 45 ml QFF column. 

Wash with 100 ml 50 mM NaCl, then 50 ml 100 mM NaCl 

Elute with 200~400 mM NaCl. Protein started to elute at 150 mM&nbsp; but the peak is tailing if 150 mM NaCl is used. 

Should use 100 to 500 mM linear gradient. 

[[Image:T4 DNA ligase purification Q FF lot 20121223.jpg]] 

This is another run using 4 5 ml Q FF in tandem. Looks like Q FF column is not good 

[[Image:T4 DNA purification Q FF lot 20121223.jpg]] 

<br> 

So I concentrated protein from Q FF column using Pierce 9k spin column, load 4 ml to BioRad BioSil 

TSK Gel 250 column. Run gel filtration. The peak is quite nice, but the protein is still not as pure as previous lot 

The purity is about 90%. After dialysis vs 50% glycerol , I got ~ 3 ml, 13 mg/ml. I labeled it as 12 mg/ml 

This gel shows the purity. 

[[Image:T4 DNA ligase QC Lot 20121223.jpg]] 

<br>