Hello,
I'm working with clinical isolates of A.Fumigatus for my secretome analysis. Previously, I used Millipore ultrafiltration membrane in amicon ultrafiltration unit to concentrate the large volume of protein sample. Now, I'm in sort of membrane to pursue my work further. I would like to get suggestions for the alternate method to concentrate the large volume of protein sample.
Also,
1. Can I use the same membrane for different strain?
2. How many times can I use the membrane?
Thanks in advance friends.
1. Yes, as long as the size of strain sample is larger than poresize of membrane.
2. Membrane can be used to concentrate the protein sample continuously
Hi Suresh,
Hard to comment on specifics, but...
Have a look at the different specifications of the membranes - some are good for several uses, others not. There's also membranes that filter out different protein sizes (
Hi Suresh,
Ultrafiltration is a good method to concentrate proteins, since it allows to you reuse them. If you have a large volume to filtrate you should increase the membrane area. But assuming that you don't have budget to buy other system and that you don't recovery the proteins, I would say that the addition of acid followed by centrifuge is a good method.
1. Concerning to use the same membrane, it's up to you. If contamination is critical, I suggest to change the membrane (since membranes are not expensive). But if you don't have budget enough, you can make a basic cleaning, followed by sodium bisulfite cleaning (for disinfection). I
2. Concerning the second question, you have to compare the initial water permeability with the permeability after cleaning. If increases more than 10%, your membrane is damaged. IReplace it.
How big is big volume and what do you intend to do with the consetrated samples?
Alternative concetration approaches:
80% Amonuum Sulfate - diaysis recomended for activity.
Acetone percepitation - sometimes destructive activity wise
TCA or TCA/Acetone - good for SDS-PAGE but destructive activity wise.
Probebly a few more. Again, it depended on the next step.
Yoram
Hi Suresh,
If you have to process large volumes, I would say to increase the membrane area. But if you have enough budget, I suggest you to buy a ceramic membranes system. For clinical applications where contamination is critical they are the best option. The filtration is tangential (so you minimize the membrane fouling) , you can clean it without damage and sterilize by autoclave. The big advantage is that a ceramic membrane can be reused during 2 years or more, without any problem.
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