Protein purification questions - (Feb/27/2019 )
1) What is meant by a weight ratio? I have been told to add TEV in a weight ratio of 3:1, substrate: cleavage factor. An example would be very appreciated.
2) What is the protocol for batch purification to remove TEV protease via the use of NI-NTA agarose beads?
1) Weight ratios mean exactly that a ratio based on weight (mass) - if you have 1 mg of substrate, add 3 mg of TEV.
2) I suggest you google: on-column protease digestion his-tag
So say I have 2ml of 2mg/ml of my substrate and my TEV is 4.5mg/ml. So I have 4mg of substrate in total. My ratio is 3:1 so 4mg:1.3mg TEV. To get 1.3mg TEV I would use 0.29ml. So my final solution would be 2ml of my 2mg/ml substrate and 0.29ml of my 4.5mg/ml TEV? Is that correct?
Sounds about right. I see in my post above I got the ratios around the wrong way, so you even managed to interpret what I intended correctly.