Hi every body! i am trying to pool the protein concentration after removal of his tag from native protein for crystal but did not get required quantity. When I expressed, purified and concentrated this protein at small scale , i got 18ul/mg concentration. Also when I tried to express, purified and concentrated at large scale I only got 6ul/mg. Kindly suggest to me what i should to do?
It sounds like a scaling issue. The simple answer is to do the purification and concentration step in small batches and then pool them.
If you need to scale up; check that you mixing is still efficient (you'd be amazed at the difference mixing makes), look at your filtration methods (you can loose lots of material to the filters), and if you are using exchange membranes, look at the volume to surface area ratio's (you'll find that as the diameter of your tubing increases, the volume increases at a greater rate than the surface area).
Hi there,
It is usually said that 10mg/mL is the required protein concentration for crystallization trials. But actually the total amount of protein for the first round is depending on the equipment you have. If working at 100nL per drop, a few tens of µL of 10mg/mL will be enough to prepare hundreds of drops . So actually the limiting factor is how much protein you have and how does it behave during the concentration process. To come ack to your question what are the volumes and concentrations of the purified proteins?
Sorry to say all of you . I made mistake in written statement it was not 18ul/mg but it was 18mg/ml. I am using Amicon Ultra-15 10K Centrifugal filter device (Millipore) for the pool of protein concentration . This device was broken i didnot get notice that's why I have lost my protein . Now i got 30 mg/ml protein in concentration.Thanks for you suggestions and support.
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