Hello all, I am liitle confused on reusing my resing after purification of protein using gravity flow through column. I am using cobalt resins and I am doin an His-tag purification. So I incubate my protein with cobalt resin overnight at 4 degree and proceed with further process for purification. my questions are.
1) Is it alright to reuse the resing in the column for purifying and alternative protein which is HIS tagged.
2) If yes, do I have to go through the process of incubating my alternative protein over night at 4 degree or is it alright to load my clear lysate for purification process with out over night incubation after equiliberating the raisins with resuspension buffer
Thank you
The charged resin can be used multiple times (5 to 6 times or as given in the manual) before recharging again for the purification of same protein . It is recommended to use separate resin or prepacked column for different his tagged proteins.
You can always run a washing procedure if you suspect protein precipitation. Incubation overnight is just necessary when the tag is somewhat hidden or you have strong diffusion resistance.
1 hour incubation of the protein with unpacked and equilibrated resin in a beaker or a falcon is more than sufficient for binding, you don't have to incubate for that long (overnight).
For a packed column the protein can be loaded employing a slow flow rate
Question 1) It is better do not use the same column for different proteins. However (if you have only one column), you can clean following the instructions and reuse as many times as you can, but perhaps after many times the cobalt charge decreases and you would need to regenerate the column.
Question 2) Of course the time depends of the sample volume (e.g. are 500 ml need much more time than 5 ml) and the binding capacity of your resin. As Dr Singh wrote 1 hour of incubation is enough for a first approach if you work in batch. If you can use a Low pressure system or an FPLC, try to apply your sample at 0,5-1 ml/min until Absorbance goes to Zero. After this, you can try to elute your protein using a linear gradient of imizadole.
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