Microsomal preparations from ram seminal vesicles. Ram seminal vesicles are obtained at slaughter and immediately frozen at -70C. These vesicles are an excellent source for isolation of several eicosanoid metabolizing enzymes. These microsomal fractions are useful for enzyme purification or by addition of inhibitors, as a crude source of specific enzyme activities. Each vial contains 5mg, which is sufficient for approximately 100 oxygraph assays. Eicosanoid metabolizing enzymes such as cycloxygenases, prostaglandins, and numerous other enzymes, are microsomal membrane associated and are found highly expressed in seminal vesicle tissues. This microsomal fraction has been isolated from ram seminal vesicles with care to minimize the loss of enzymatic activity. Source: Microsomal fractions were derived from frozen ram seminal vesicles that were collected and placed at -80C immediately after excision from post-slaughtered male ovine bucks. Enzyme Activity: 169pmol of metabolized Arachidonic Acid/ug protein/min at 37C 0.5pmol. This microsomal fractionation can be used as a source of active eicosanoid metabolizing enzymes. Indomethacin Inhibition: This preparation of microsomes exhibited and IC50 of ~55nM for enzyme inhibition of Arachidonic Acid with Indomethacin. Applications: Suitable for use in Polyacrylamide Gel Electrophoresis/Western Blot. 20ul is sufficient for mini-gels. Larger gels will require more sample to be loaded. Concentration: 5mg/ml total protein using the BCA protein assay with BSA as a standard. Solubilization Protocol: Many eicosanoids can be solubilized using the following protocol: NOTE - All steps should be performed on ice, using ice cold solutions, or at 4C where appropriate. 1. Ultracentrifuge suspended microsomes at 110,000 x g for 90 minutes and discard the resulting supernatant. 2. Resuspend microsome pellet in 2-4 x volume of 10mM Tris-HCl, 0.5mM EDTA, 1% Tween 20, pH to 8.0 using deoxygenated water with 3X strokes of a Teflon pestle homogenizer. 3. Stir suspension for 45 minutes. 4. Ultracentrifuge the stirred suspension of microsomes at 100,000 x g for 90 minutes and collect the supernatant of solubilized eicosanoids. The solubilized eicosanoids are now ready for further processing or aliquot and storage at -80C. Storage and Stability: Stable for at least one year at -20C and indefinitely at -80C.
Reinheit:
Protein suspended in SDS/mercaptoethanol sample buffer with a bromphenol blue dye marker.
Formulierung:
Supplied as a frozen liquid in 100mM sodium phosphate, 250mM mannitol, 1mM PMSF, 10mM EDTA, pH 7.8
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