Buffer composition is 50mM tris pH7.5(p.I 6) 150mM Nacl (tried upto 300mM) 5mM BME 1mM pmsf 10% glycerol. Even it is also aggregating after NI -NTA purification but I am diluting protein then it is ok but during concentration it is aggregating.
Hello Ram, these spin UF filters are great, but one has to be a little cautious about using them, especially for the first time. Some proteins have a tendency to bind to the membrane, and even if the binding is reversible, reducing the environment of the protein molecule from 3D to 2dimensions can increase the chance of a protein-protein aggregation event. A few points for you to consider:
1) What is your starting protein concentration? If it is low, you could be loosing material on the membrane. Check this if you can.
2) What is your membrane composition/chemistry? Sometimes, trying different types of membranes can help a lot. If you are using a polysulphone or polyethersulphone, it may be worth looking at regenerated cellulose, for instance. Different chemistry may be 'kinder' to your protein.
3) Use as few spin concentrators as possible. It is tempting to run 2, 3 or more in parallel to speed up the concentration, but this can lead to more loss and more problems.
Of course, it could be that your protein has a tendency to aggregate regardless of the membrane concentrator, once a certain protein concentration is reached. You may want to consider whether detergents such as 0.1% Tween20 or TritonX100 can be tolerated in your application(s) and try adding them, as they can also help.
Hope this helps, Dave
If your protein is an inclusion one it may aggregate. Check whether it is soluble protein or inclusion protein. Also check about column (I think amicon columns are lined with membrane).
Hello Ram, these spin UF filters are great, but one has to be a little cautious about using them, especially for the first time. Some proteins have a tendency to bind to the membrane, and even if the binding is reversible, reducing the environment of the protein molecule from 3D to 2dimensions can increase the chance of a protein-protein aggregation event. A few points for you to consider:
1) What is your starting protein concentration? If it is low, you could be loosing material on the membrane. Check this if you can.
2) What is your membrane composition/chemistry? Sometimes, trying different types of membranes can help a lot. If you are using a polysulphone or polyethersulphone, it may be worth looking at regenerated cellulose, for instance. Different chemistry may be 'kinder' to your protein.
3) Use as few spin concentrators as possible. It is tempting to run 2, 3 or more in parallel to speed up the concentration, but this can lead to more loss and more problems.
Of course, it could be that your protein has a tendency to aggregate regardless of the membrane concentrator, once a certain protein concentration is reached. You may want to consider whether detergents such as 0.1% Tween20 or TritonX100 can be tolerated in your application(s) and try adding them, as they can also help.
Hope this helps, Dave
try diluting the buffer composition before loading in to the amicon concentrator. Some chemicals were nt found to be compatible with these filters. usually we have to use 15ml in the uppper portion. EX: if ur samples is 5 ml (1x buffer) add 10 ml (0.1X buffer).
Hi,
Check your critical micelle concentration (CMC) cut-off values in your buffer. Also, refer the data sheet of amicon filters. http://www.millipore.com/userguides/files/centrifugal/$file/PR03520TR_RevA_English.pdf.
Raja Sekhar
Aggregation during concentration can be many reasons, e.g., instability of your protein. It is not bad that the aggregation is reversible. To first exclude the possibility that the aggregation is caused by concentrating itself: 1. try to pipette the protein up and down every 5 to 8 minutes and prevent increasing regional hydrophobic interactions. 2. try vivaspin instead of using Amicon.
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