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Hi guys,
I have cloned a 500bp gene into pET21b and after expression I found out that 3 bands are expressed and after purification it shows 3 bands as well as western blot result. one band is exactly the same as desired protein and the others with lower molecular weights (about 2-5kDa differences). I should point out that I double digested the PCR amplified insert using NdeI and HindIII and the strange thing was that on gel agarose it seems that the digested bands are aggregated and resulted in very heavy band! I incubated them at 50degree and after that it results in 2 bands (one the same as desired and the other heavier). I am not sure if the enzymes are working insufficiently.. and so result in different bands or just the problem is in expression part.
Can anyone give me a suggestion?
