the ELISA are for chemo/cytokines. Manufacturer sent me some papers but not finding anything I feel confident using.
Dear Jane,
We have used the following homogenization buffer in procedure for TNF-alpha concentration determination in homogenates of rat livers: Animals were killed and livers were rapidly excised, rinsed of blood and homogenized by polytron (Brinkman) in homogenization buffer (PBS containing 0.05% sodium azide, 0.5% Triton X-100, and a protease inhibitor cocktail, pH 7.2, 4˚C) and then sonicated for 10 minutes. Homogenates were centrifuged at 12,000 x g for 10 minutes, and TNF-alpha amounts in the supernatants were determined by ELISA. The TNF-alpha content was expressed as pg/mg total protein. Total protein was determined by the Biorad assay. Also we started using KRC3012 with liver tissue homogenates, according to the paper of Borovikova et al. in Nature 405: 458-462 (2000).
Also, to determine IL-6, we used the following procedure for the homogenization of rat liver. The liver was sliced into sections of about 500 mg on dry ice and them placed in 2 mL of ice cold phosphate buffered saline containing 13 microliters/mL of protease inhibitor (Sigma catalog number P8340) plus 5% fetal calf serum. We also used KRC0062 with serum and with tissue homogenates. The tissue was homogenized on ice and then was diluted to 5 mL with the same solution. The homogenate was then centrifuged at 1500 xg for 15 minutes. The clarified supernatant was then apportioned into 1 mL aliquots and stored at –70 degrees C until ready for use.
Best wishes,
Ilya
In my experience with ELISA for tissue derived cytokines, it is better to avoid contamination of blood components in the tissue which you want to analyze. While it takes bit of time (1-2 minutes) to flush through portal vein, it is worth it. Recipes are less important, but I prefer to use Tris based lysis buffer with NP-40.
1- Liver tissue its very heterogeneous cell types, first you have to do many wash with saline as well as PBS buffer to remove the RBC,s and blood cells. Then, it is better to make a single cell suspension first and extract as regular way for single cell extraction.
I ended up using a commercial buffer, T-PER (Thermo Fisher Scientific 78510), which worked pretty well. I used a bead system for homogenization as I had a ton of samples. Thanks everyone for your help.
An alternative to everyone in the future:
https://en.cellsignal.de/products/buffers-dyes/cell-lysis-buffer-10x/9803
Does homogenization buffer for liver tissue Elisa differ from Lysis buffer for liver tissue protein extraction?
Probably not, lysis refers to breaking up the cell, and homogenization means you are breaking up the tissue, but the cells will get lysed in this case as well. The terms tend to get used interchangeably but I like to use homogenization for tissue and lysis for cells only.
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