The overall goal of this procedure is to show the synthesis of activity based probe, ARN 14 six eight six specific for N-acylethanolamine acid amidase, or NAAA, and describe its use for detecting the catalytically active enzyme in cell extracts and tissue sections. This method can help answer questions about the in viva function of NAAA providing insights into NAAA activation in different physiological and pathological conditions such as inflammatory and neuro degenerative diseases. It's main advantage is that a simple manipulation is subsequent to NAAA reaction with a probe thus giving reliable information about the in viva activation state of the enzyme.
Another advantage is that contrary to specific antibodies our probe detects only the active form of the enzyme which is useful for fluorescence microscopy status. Visual demonstration of this method is critical as it combines different expertise that built up to a complete story from proper synthesis to its final applications. In a 50 milliliter round bottom flask dissolve 350 milligrams of undecynenol and three point five milliliters of dry dichloromethane.
Then add 25 milligrams of DMAP and 530 milligrams of DPC to this solution. Stir the mixture at room temperature for 16 hours. After 16 hours add 20 milliliters of dichloromethane to the solution.
Transfer the mixture into a separating funnel. Add 15 milliliters of water. Shake the mixture and allow the two phases to separate.
Next open the stopcock, collect the organic phase on the bottom close the stopcock and transfer the organic phase back to the funnel. Add 15 milliliters of a saturated sodium bicarbonate solution to the mixture. Shake the separating funnel and let the two phases separate.
After drying the organic layer with sodium sulfate filter it through cotton into a tiered round bottom flask. Then evaporate the organic layer to dryness under reduced pressure using a rotary evaporator. Weigh the flask and obtain 600 milligrams of oil which is a mixture of two pyridyl undecynyl carbonate and undecynol two oxypyridine carboxylate in a ratio of one point seven to one.
In a 10 milliliter round bottom flask dissolve 60 milligrams of oxo asetydenol ammonium asetate in two milliliters of dry dichloromethane. Cool the solution to zero degrees celsius in an ice bath then add 81 microliters of diazapropalethylamine drop wise. Next dissolve 350 milligrams of the crude mixture containing undecynol two oxypyridine carboxylate in two milliliters of dry dichloromethane.
Add the dissolved mixture to the solution and stir at room temperature for 15 hours. Then evaporate the solvent to dryness under reduced pressure using a rotary evaporator. Purify the compound by silica gel column chromatography using an automated column chromatography apparatus.
First absorb the sample onto silica gel. After equilibriating the column with cyclohexene load the sample into the cartridge. Elute the sample with ethyl acetate in cyclohexane from zero to 100, collecting the peaks into test tubes.
Evaporate the solvent of the fractions corresponding to the compound to dryness under reduced pressure using a rotary evaporator to obtain 40 milligrams of a white solid. After pre clearing the sample as described in the text protocol add 11 point three microliters of a five millimolar stock of azide polyethylene glyco biotin and vortex. Then add 11 point three microliters of a 50 millimolar stock of freshly prepared TCEP to the sample and vortex.
After premixing 34 microliters of a freshly prepared working solution of one point seven millimolar TBTA with 11 point three microliters of a 50 millimolar copper sulfate stock add 45 point three milliliters of this solution to the sample and vortex. Use labeled containers. Incubate the reaction at 25 degrees Celsius for at least two hours.
After one hour incubation observe protein precipitation and mix the reaction. Centrifuge the sample for four minutes at 6, 500 times G at four degrees Celsius. After removing the supernatant add 750 microliters of cold methanol and resuspend the pellet by sonication using a probe sonicator for five seconds.
Then centrifuge the sample for four minutes at 6, 500 times G at four degrees Celsius. And remove the supernatant with a syringe and needle. After washing two more times with cold methanol add 325 microliters of two point five percent SDS in PBS to the protein pellet.
Sonicate the sample three times for five seconds. Next heat the sample for five minutes at 65 degrees Celsius. After sonicating centrifuge the sample for five minutes at 6, 500 times G at room temperature.
Then transfer the supernatant to a clean 15 milliliter tube and add one point four milliliters of PBS to the supernatant to dilute the SDS concentration to zero point five percent. For streptavidin enrichment first bring the volume of the sample to four point two milliliters with PBS. Then add 40 microliters of a 50%slurry of prewashed streptavidin agarose using a cut end tip.
Incubate the mixture for two hours at room temperature with rotation. After centrifuging for two minutes at 1, 400 times G remove the supernatant without drying the bead pellet. Use the residual supernatant to transfer the beads to a one milliliter spin column and wash the column by gravity with one milliliter of one percent SDS in PBS.
Continue washing the beads with six molar urea followed by PBS as described in the text protocol. Then use 500 microliters of PBS to transfer the washed beads to a one point five milliliter tube. After centrifuging the sample for two minutes at 1, 400 times G gently aspirate the supernatant with a syringe and needle.
To elute the resin bound proteins add 25 microliters of elution buffer to the resin pellet and incubate for 15 minutes at room temperature followed by 15 minutes at 95 degrees Celsius. Add approximately 400 microliters of click chemistry mix to tissue slices which have been prepared according to the text protocol. Pay attention that the chosen volume of click chemistry mix is sufficient to cover the slices.
Then incubate the tissue slices for one hour at room temperature protected from light. Following incubation wash the samples with PBS for five minutes and then with cold methanol for five minutes. Next wash the tissue slices three times with a solution of one percent Tween 20 and zero point five millimolar EDTA in PBS for two minutes.
Finally wash the slices one more time with PBS for five minutes. After air drying add a drop of antifade mountant with DAPI. Close the samples with cover slips and seal with polish.
For probe synthesis undecynenol is activated by dipirydyl carbonate in the presence of catalytic four dimethyl aminyl pyridine to produce a mixed carbonate which is then reacted with the aminyl lactam the beta lactam or head of the probe reacts with the NAAA active site while the terminal alkyne can be functionalized with a reporter tag via click chemistry. ARN 14 six eight six was used to detect NAAA activation in cell extracts of rat inflamed paws by protein block. In naive rats, no NAAA signal is detected.
Conversely in rats treated with complete Freunds Adjuvant a marked band corresponding to active NAAA is observed. Another application of ARN 14 six eight six is tissue imaging by fluorescence microscopy. Here red cells represent alveolar macrophages which are positive for active NAAA.
NAAA is localized in diffused vesicular structures as appreciable at a higher magnification. After watching this video you should have a good understanding of how to synthesize and use ARN 14 six eight six to detect active NAAA in tissues both by protein blot and fluorescence microscopy. In addition even though this protocol is specific for our target, the probe applications that we describe would be easily adjusted for different activity based probes specific for targets other than NAAA.
