I unmindfully added 1X TBST in place of washing buffer to the Ni-NTA column after the flowthrough during protein purification. Though I didn't allow the beads to incubate with the buffer, I immediately decanted the column and washed it three times with the wash buffer. Will the 1X TBST affect the Ni beads or the bound protein? I continued my elutions and washed the column with wash buffer and autoclaved water afterward. Do I need to recharge the Ni-NTA column before the next purification or need to add fresh beads?
I do not think that the beads will be affected, the separation process might though. With respect to the protein, you can expect to have tween-20 bound to it, through hydrophobic interaction. On the other hand, washing and Ni ions recharging should be performed always to obtain reproducible results (there is always Ni ions leakage due to the adsorption process and some nonspecific binding).
Hi Sampurna,
I agree with Omar Gonzalez-Ortega . It's not a major issue. Simply recharge the Ni-NTA beads and wash them thoroughly several times; they should be ready for reuse.
Thank you for the reply, observed the expected band post SDS gel run and have also recharged the beads for future elutions.
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