I'm trying to purify the TCF3 protein (~70-75kda kDa, pI 4.5) from E. coli BL21 cells after inducing expression with 1 mM IPTG. I am using Ni-NTA affinity chromatography to purify the His-tagged protein (C-terminal His-tag), washing with 30 mM imidazole and eluting with 250 mM imidazole. However, I am observing the presence of non-specific E. coli proteins in my elution fractions.
When I attempted purification using cobalt affinity resin instead, I did not detect any protein in the elution or wash fractions, but it appeared in the flowthrough instead. Why might this be happening, and how can I improve the purification to obtain a cleaner TCF3 sample?
Hi,
Try using a gradient of imidazole in your elution buffer to improve purity. Start by washing the column with 50 mM imidazole, then gradually increase the concentration in steps of 50 mM (i.e., 100 mM, 150 mM, 200 mM, 300 mM, and finally 500 mM). This stepwise elution helps remove weakly bound contaminants while ensuring your His-tagged protein elutes efficiently.
Adding 0.3 M NaCl to the buffer can also help by reducing non-specific electrostatic interactions, improving the specificity of binding to the column.
For further purification, consider using size-exclusion chromatography (SEC) to separate your target protein from any remaining impurities. Finally, check the purity of your fractions using an SDS-PAGE gel
- Badges
- Science topic
- Similar topics
- Control Systems Engineering
- Filtering