Hi, I´m trying to extract protein from mouse lung for WB, using TR3 lysis buffer consisting of glycerol, SDS and Na2HPO4, but I´m facing a problem at the end step when I try to take the supernatant, I couldn´t because of the mucus formed and I got little yield of supernatant? anyone have experience so can give me some hints?
try a mechanical dissociation step first to get rid of the mucus before adding any SDS. Try mincing with razor blades and passing chunks through decreasing needle calibers (16G-->22G) in a syringe. Also consider using a better buffer than Phosphate, try TRIS or any of the zwitterionic buffers, include proteinase inhibitors, you might include a good DNase in the mincing step, make sure not from pancreas as it might contain traces of trypsin or other proteases. On the other hand, if the mincing is quick, traces of proteinases might be a good thing. After mincing, wash the chunks in PBS or saline and you'll notice that the mucus due to lower density will float. - Good luck.
Hi Khadija, I have not tried to extract proteins from lung, but I extracted proteins from liver, spleen, kidney, heart and different cerebral areas. Maybe your problem is caused by nucleic acid, I do not know. I usually use scissors and Dounce tissue grinders (loose and tight) to destroy the tissue in lysis buffer (containing protease inhibitors and nuclease) with ice bath. After that the lysis solution will be incubated for half an hour. Centrifuge and get the supernatant. Thanks.
SDS tends to liberate copious amounts of viscous genomic DNA from almost any tissue. I use home-made Laemmli's buffer (2% SDS, Tris, Glycerol), and I've found that I need to sonicate my samples to shear the DNA before I can really pipette the supernatant. On the upside, sonication also increases protein yield.
I usually homogenize in ice-cold PBS (or sterile saline), take an aliquot and mix 1:1 with 2X RIPA buffer with protease and phosphatase inhibitors, sonicate and spin at low speed (0.8 rpm for 5 min at 4C). You'll get a clear supernatant with a lot of protein that you can use for downstream applications such as WB.
Thank you very much for your answers. Just to clarify I tried to use a bladde to homogenize the tissue which was the lung but I got this mucus after adding the cell lysis buffer plus protease inhibitor and PMSF, so the next time I tried grinding the lung with the mortar and pestle in liquid nitrogen, and I got also this mucus again.
It could be usefull as some of you said to use DNase and maybe to sonicate then centrifuge to collect the supernatant.
I've considered trying DNAse/RNAse or benzonase, but I think the SDS will inhibit the enzymes, hence why I've stuck with sonication. If you find that nuclease treatment does work please let me/us know.
have you tried washing the mucus out before you start, by infusing through a large airway?
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