Hi..I have over-expressed a protein (tagged with His) via retroviral transduction method in THP-1 cells. Now I am interested to check if there is any binding partner with my tagged protein in a particular condition. I intend to pull down my tagged protein using Ni-NTA resins so that any interacting partner may come with the tagged protein. Subsequently I planned to perform Mass-spect to identify it.
Can you please help to find out a suitable centrifuge based protocol for the pull down experiment?
Thanks in advance.
As stated by Kariona purifying with imac can be tricky. Try the mentioned procedure and run sds-page for several fractions to determine purity of the desired protein.
Thank you Kariona mam for your detailed answer and thanks to Omar sir also.
Just an experimental suggestion...Ni-NTA based protein purification gives lots of non-specific bands,,at least that is what I have seen in bacterial system. So, use 10 mM imidazole in your binding buffer, incubate with resin preferably for not more than 2-3 hours (depending on your case), wash it three times with wash buffer containing 20 mM imidazole (you can play with that depending on your proportion of specific vs non-specific bands and their respective affinity towards the bead) and finally elute it with elution buffer containing ~ 200mM imidazole. The concentration of imidazole can be standardized. Collect all the fractions, even the unbound/washed fraction, load all fractions on a gel to get a detailed picture of your experiment. If you are suspecting any specific protein that can interact with the tagged protein, you can simply do a western blot and if you want to know the whole interactome then go for mass spec as mentioned by you. All the best.
Hi there,
To avoid non specific and low affinity interactions, you may preclear your lysate by treating it with naked beads (without Ni++ immobilized ion) and then apply systematically an imidazole concentration which will limit the low affinity interactions. As a control you may run an experiment with the cell line overepressing the non tagged protein.
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