What Is The Separation Of Cockroach Ether

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PURIFICATION OF CRUDE COCKROACH EXTRACT MATERIALS REQUIRED: • Cockroach- Periplaneta americana • Mortar and pestle • Diethyl ether • Lyophilizer PROCEDURE: • Cockroaches maintained at -700C were lyophilized. • The freeze dried cockroaches were ground to powder with mortar and pestle. • The powder was dried cockroach powder. • De-fatting the dried cockroach powder. 1. Diethyl ether (4-5 times) was added to powdered material and was kept at 40C on magnetic stirrer. 2. Diethyl ether solution was changed after every 3 hours till clear layer was obtained. 3. The ether was drained off and the powder was dried and then stored in a cool and dry place. EXTRACTION REAGENTS AND BUFFERS: 1. PBS-extraction buffer (for 1000 ml) • Sodium chloride -8.8g • Sodium dihydrogen phosphate(NaH2PO4) -3.585 g • Disodium hydrogen phosphate(Na2HPO4) -11.78 g 2. PMSF stock (for 100 mM) • Is prepared in isopropanol and stored at -200C • Final concentration used is 1mM 3. EDTA stock (0.5M) It is insoluble in water and dissolve only when neutralised with NaOH in pH=8. MATERIALS: • Extraction buffer • PMSF stock (100 mM) • EDTA stock (0.5 M) • Beaker • Magnetic stirrer • Centrifuge • Whattman Filter Paper PROCEDURE: • Deffated cockroach powder (2g) was weighed • To extraction buffer (98 ml, stirred continuously), add I ml of 100 mM of PMSF (final concentration is 1 mM) and 1ml 0.5 M of EDTA (final concentration of 5mM). to it 2 g of de-fatted cockroach powder was added. • The beaker was covered with the foil and stirred constantly on magnetic stirrer overnight (ideally 8 hrs) • After overnight extraction magnetic bead was removed and the extraction buffer was centrifuge... ... middle of paper ... (TMB) reagent • Stop solution 1. 2 N sulphuric acid • NUNC flat base ELISA wells PROCEDURE: • Prepare capture antibody solution of dilution 1:250 in coating buffer and add 100 µl in each well and incubate at 40C overnight. • Next morning aspirate and wash the wells 3 times with PBST • After washing add 200 µl blocking solution which is 10 % FBS in PBS in each well and incubate for 1 hour. • Aspirate and wash 3 times. • Add 100 µl standard and samples to the allotted wells. • Incubate overnight at 40C. • Aspirate and wash 5 times with PBST • Add 100 µl of secondary plus detection to each well and incubate at room temperature for 2 hours. • Aspirate and wash 7 times with PBST. • Add 100 µl of substrate solution to each well and allow it to develop for 30 minutes in dark. • Add 50 µl of stop solution to each well and take the readings at 450 nm within 30 minutes.
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