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[shinebio]

Hi,
I am trying to express membrane protein with Pichia. I got the proteins in the first expression, but somehow I can't get them anymore with the same protocol.....
My protein binds a pigment that I can see clearly if it is expressed when I spin down the cell pellet by the color(red). But now, after breaking the cells with microbeads in the sodium phosphate buffer at pH 6.8, I notice the redness fading away and I can't only get the dark and dull colored membrane after ultracentrifugation...I found there are some black "things" at the rim of the pellet after I broke the cells and spin at low speed to get rid of the unbroken cells.....does it mean that it is overheated during the cell disruption and the cells get burnt?