I 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

General recommendations

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.

also i would like you to pay attention to cross creativity of the kit with other prostaglandin compounds (PGE1, PGE3, PGF1α, PGF2α, 6-keto-PGF1α, PGA2, PGB1, 13,14-dihydro-15-keto-PGF2α..........etc)

good luck 

Hi Barbara,

I am familiar with our PEG2 Sandwich EIA kit (SKT-207). We outline specific handling instructions for the various samples types, specific for our kit.

I would recommend that you ask the supplier of the kit for their guidelines, as they will vary between kits.

Best of luck!

http://www.stressmarq.com/Products/Kits/SKT-207.aspx

Dear Barbara,

You don't have to make any sample purification.  The kit is particularly selective for PGE2 as it includes monoclonal antibodies.

Good luck

Thank you Leslie Rietveld and Ashraf Sayour !!!

You are welcome, Barbara.

I 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

General recommendations

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.

also i would like you to pay attention to cross creativity of the kit with other prostaglandin compounds (PGE1, PGE3, PGF1α, PGF2α, 6-keto-PGF1α, PGA2, PGB1, 13,14-dihydro-15-keto-PGF2α..........etc)

good luck 

Thank you Ashraf !