For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome
If you continue without changing your cookie settings, we'll assume you’re happy with this.
I have kit (ab133021) prostaglandin E2 elisa kit and I did not find the procedure of tissue homogenate in the instruction
Asked on Apr 22 2015
We would recommend to prepare tissue homogenates using a standard method as the one described below:
Dissect the tissue of interest with clean tools, on ice preferably and as quickly as possible to prevent degradation by proteases.
Place the tissue in round bottom microfuge tubes and immerse in liquid nitrogen to "snap freeze". Store samples at -80°C for later use or keep on ice for immediate homogenization.
For a ˜5 mg piece of tissue, add ˜300 µl complete extraction buffer (see cell/tissue extraction buffer recipe below) to the tube and homogenize with an electric homogenizer.
Rinse the blade twice using 300 µl complete extraction buffer for each rinse, then maintain constant agitation for 2 hr at 4°C (e.g. place on an orbital shaker in the cold room).
Centrifuge for 20 min at 13,000 rpm at 4°C. Place on ice, aliquot supernatant (this is the soluble protein extract) to a fresh, chilled tube and store samples at -80°C. Minimize freeze/thaw cycles.
Volumes of lysis buffer must be determined in relation to the amount of tissue present. Typical concentration of final protein extract is >1 mg/ml.
Cell/tissue extraction buffer recipe
100 mM Tris, pH 7.4
150 mM NaCl
1 mM EGTA
1 mM EDTA
1% Triton X-100
0.5% Sodium deoxycholate
Recommended protein extract concentration is at least 1-2 mg/ml.
Typically, serum, plasma, cell and tissue extracts are diluted by 50%.
Prior to use after thawing, centrifuge samples at 10,000 rpm for 5' at 4°C to remove any precipitate.
As explained on page 11 of the protocol booklet, in order to help preserve the PGE2, we suggest the immediate addition of prostaglandin synthetase inhibitors such as indomethacin or meclofenamic acid at concentrations up to 10 μg/mL should be added to either the tissue homogenate or urine and plasma samples.
Guillaume BoucherAbcam Scientific Support
Answered on Apr 22 2015