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Assuming you followed the above advice and used an appropriate binding buffer and your protein isnt huge, then a likely reason for this is that the expression yield is much lower than you expected

Exercise can increase serotonin and dopamine levels in the brain, similar to the effects of estrogen, providing a natural way to mitigate some of the cognitive and emotional symptoms of menopause [102]

Annals of the New York Academy of Science 890, 167-172CrossRefGoogle ScholarPubMed 55Ferris, C.D., Jaffrey, S.R., Sawa, A

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References Martinvalet, Denis & Michael, Walch