Hi,
I'm using Hitrap SP FF collumn (cation exchange collums) to purifiy PDGF. The collumn's details show binding capacity of some proteins:
70 mg Ribonuclease A/ml gel (Mw: 13 700 D)
50 mg human IgG/ml gel (Mw: 160 000 D)
50 mg Bovine COHb/ml gel (Mw: 69 000 D)
Therefore, how much amount of samples can I apply maximum into Hitrap SP FF collumn? How can I configure out that amounts?
*Collumn's volume is 5ml
Thanks for you help!
Regards,
Trung,
What is the pH that you want to work with? When calculating how much protein to add to your column, you have to remember that not all protein will bind to it. Some people will tell you that you should not overload a column and they are indeed right. But, if you have tons of starting material and do not mind loosing some PDGF I would load 5 X 70 mg of total protein to your resin. You should be careful in assaying your flow through to ensure that you did not loose some of your PDGF. This way you will have more purified PDGF when you are done.
Hope this helps,
Marc
It depends on the column size, it should be 1/6th of void volume.
Thank you, Marc.
The sample is applied with pH 4.0 . I see that amount of protein binding onto collumn don't depend on molecular weight. For example, IgG (160 kDa) has the same volumes with Bovinine (69kDa) or even Ribonuclease A (13.7kDa). Therefore, I wonder how can I configure out amount of PDGF which can be applied. It will be a basis for me to design experiment.
Thanks, Ravi.
However, I don't know the capacity available of collumn for PDGF. I'm finding the answer.
Trung,
I think your pH is somewhat low, do you know the pI of your protein? Rule of thumb is that you need to be minimally 0.5 pH unit away from the pI. Going too far from the pI may damage the protein. You are correct ion exchange depends on total charge and not size of a protein. Also by the way I loaded liters of very dilute sample onto an ion exchange, I do not see how the void volume is important since this is a capture method.
hope this helps,
Marc
Marc,
The pI of my protein is 9.8. According what you say, it's seem damage the protein. This thing I don't know, I never read in any paper.
I'm calculating amount of my protein base on electrostatic surface. I think the total charge between two samples is equal. Therefore, amount of my sample is (70/13.7*29)/24*30 = 179.1 (mg) (maximum amount of Ribonuclease A (13.7kDa) is 70mg. The total charge of Ribonuclease A and PDGF are 29e and 24e, respectively)
Do you think it right or wrong?
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