I would like to make protein lysates from rat intestine to measure cytokines in the profile. Does anybody have a good protocol how to do that? I already found in the literature that intestine might have a lot of proteases and a special inhibitor cocktail might be needed.
Thanks for your help!
Isabell
Roche has a series of complete protease inhibitor mix tablets; they're great and super simple to use. Also, proteases tend to be a lot less active at 4°C, so try to keep all samples and buffers on ice at all times.
We have been using CelLytic-MT (sigma) for our tissues with good success. The most important is to properly homogenize your tissue (rotorstator is great for most tissues), and be fast about it. Best to use short bursts, cooldown on ice, and repeat, rather than long homogenization times.
Good luck!
Intestine is really a collection of several very different tissues - even ascending, transverse and descending colon are different. Since you probably know what range of cytokines you're interested in, it may be worth staining the tissue to determine where they are, and then examining only that part by (say) qRT-PCR and/OR ELISA or Western blot.
Good luck!
Hi we use a bead homogenizer , MPER from Pierce and HALT from Pierce when we look at proteins and phosphoproteins. be sure to wash the tissue well and cut up into 1-2 mm pieces. Bead homogenization in short bursts in the cold room.
Dear Isabell Koxholt,
We used to mince the tissue into fine pieces and keep in -800C and polytron homogenizer (Kinematica). At the time of homogenization, we take out the samples immediately from -800C, add the lyaste buffer and homogenize it as quickly as possible before the samples get thawed. You can also keep the samples in dry ice in the intermittent period. We follow this steps to keep the tissue brittle to homogenize better. Then we allow the homogenate to 2 freeze-thaw cycles to get the lyaste for cytokine assay.
Hope this will help you
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