To compare the activity of protein in vivo

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wayne555555
wayne555555's picture
To compare the activity of protein in vivo

There is two kinds of protein that can both bind iron and form iron-sulfur cluster.But from my previous study,It seems that they have diffierent iron binding activities.So my idea is to express them in the same cell(may be E.coli) to prove that they have diffierent activity to bind iron.The difficulty is how to make sure that they keep the same expression level. Is there anyon who can give me some suggestions?Thanks in advance.

Sami Tuomivaara
Sami Tuomivaara's picture
wayne555555,

wayne555555,

Did I understand you correctly, you did _in vitro_ kinetic/equilibrium study and the proteins' iron uptake have different rate/total amount, and now you want to see if this holds _in vivo_? Could you please elaborate a bit...

I think it will extremely difficult to express two proteins at the same level, even if you use same vector and promoter. Codon usage, mRNA and protein stability, protein solubility, etc. will all affect the protein level and practically prevent you from expressing two proteins to same concentration. Your can tag the proteins with HA/His/other tag and quantify proteins later by western against your tag for normalization...

Cheers,