Protein extraction supernatant turns black/sooty color - (May/20/2008 )
I need help! I've been working with the MsrA protein in Drosophila and performing larvae protein extractions. When I extract I add a buffer, homogenize the flies with a pestle and then sonicate (keeping the sample on ice). It used to be that the supernatant would be a creamy white color and there was protein activity. Recently my extracts have been developing a black/sooty color over time and whatever it is it seems to be affecting the dabsyl I'm using for the assay. I don't know where this is coming from, all of the extractions have been done with the same strains of flies, same number of flies, same buffer, same sonicator for the same amount of time. It's weird because it seems to be some sort of oxidizing reaction because it develops at the top of the supernatant and travels down. I can even see it in the tips I use to transfer the supernatant - if there is a lot in the tip it will be black on both ends where it is in contact with the air and white in the middle... Does anyone have any idea what this could be from? I'm willing to try anything to get my samples right again.
Thank you in advanced!
jfreund1
have you checked the tip of the sonicator for pitting and/or corrosion?
i have seen solutions with black particles caused by a corroding sonicator horn.
(this may not be your problem but it is worth checking).
It's not the sonicator... Other people have used it and have not had this problem. It seems to only happen when I sonicate the larvae.
Did you use some other strain of flies too? You may have something interesting going on there with your strain.
Did you use some other strain of flies too? You may have something interesting going on there with your strain.
It's not the type of fly, it happens with different strains...