The overall goal of this procedure is to measure the activity of the e coli inducible lysine decarboxylase using a COLORMETRIC assay. This is accomplished by first reacting the substrate L lysine with the enzyme LDCI in an EOR tube, and then removing samples at three time points into a stop solution. The second step of the procedure is to react the samples in the stop solution with 2 4 6 TRI nitro benzene SUL acid or T-M-B-S-T-M-B-S reacts with the primary of the substrate and forms the products TMP lysine and TMP cadaver respectively.
The third step of the procedure is to separate the water-soluble TMP lysine from the water insoluble TMP cadaver by extracting the TMP cadaver into toluene. The final step of the procedure is to measure the absorbance at 340 nanometers of the TMP cadaver in the toluene phase. Ultimately, results can be obtained.
That CHO LDCI activity is modulated by the presence of the stringent response signaling molecules. P-P-G-P-P and P-P-P-G-P-P. Hi, I'm Hir from the atory of Dr.Wali, the Department of Biochemistry at the University of Toronto.
Today we will show you a procedure for measuring the activity of the ish e coli inducible lysine, decarboxylase, LDCI. We use this procedure in our laboratory to study the effect of novel regulators of LDCI and the acid stress response pathway of enteric bacteria. So let's get started.
To begin this procedure, prepare all necessary solutions as described in the written protocol. Next equilibrate an EINOR thermostat plus at 37 degrees Celsius. The thermostat plus has a heat block with 24 wells arranged in six columns.
Each column will be used to test a specific nucleotide, then label 24 1 0.5 milliliter einor tubes with corresponding nucleotides, fill the heat block with the labeled tubes. In addition, rack several boxes of 200 microliter tips such that each alternate row is omitted to give a total of four rows of tips. This is necessary for the use of a multi-channel pipette with a thermostat plus heat block during the enzyme assay.
Next, equate the digital heat block to 42 degrees Celsius. Place a 96, well 2.0 milliliter polypropylene plate on ice to cool. Finally, prepare a 96 well plate with stop solution.
Add 50 microliters of stop solution followed by 30 microliters of water using the VWR five to 50 microliter multi-channel pipette beacon, the LDCI assay by aliquot. 50 microliters of solution A prepared with the appropriate nucleotide into its corresponding tube. Next, add 330 microliters of solution B to three tubes in column F of the heating block.
Finally, add 330 microliters of the protein free control solution C to the final tube in column F equilibrate the solutions at 37 degrees Celsius for five minutes. Following incubation, cut off the caps of the tubes in the center of the heating block to prevent interference with the multi-channel pipette. Then use a five to 50 microliter of DWR multi-channel pipette to transfer 50 microliters from the tubes in column F to each tube in column A immediately after the first tube is mixed.
Start the timer. Continue to transfer 50 microliters of the solution from column F to columns B through E.After two minutes, remove 20 microliters from each sample using a multichannel pipette. Transfer the sample to the stop solution prepared in a 96 well polystyrene plate as described in the text.
Repeat this process at four and then six minutes after the first tube has been mixed. At this point, the samples in stop solution can be covered in plastic wrap and frozen at minus 20 degrees Celsius for subsequent processing prior to the reaction with TNBS, haw the plates out at room temperature. Then using a multi-channel pipette at 50 microliters of 10 millimolar TNBS solution to each well of the 96 well polystyrene plate containing the stop solution, incubate the plate at 42 degrees Celsius for six minutes.
The solution will turn a dark yellow orange color as the TMBS reacts with the lysine and the cadaver. After six minutes, cool the plate on ice to slow the reaction. Then remove 100 microliters of the sample using a VWR 20 to 200 microliter multi-channel pipette.
Transfer the sample to the two milliliter deep well plate that was previously cooled on ice. At this point, place the ice bucket in the fume hood. Add 500 microliters of toluene to each well, using a handy step repeat pipetter and a 12.5 milliliter pipette tip.
Using a Kim wipe, remove any excess toluene. Then cover the plate with strips of packaging tape. Be sure to press down firmly in order to obtain a good seal.
Cover the 96 well plate with a flat lid and then shake vigorously by hand for one minute and 30 seconds. Allow the solutions to settle for five minutes. The TMP cadaver is now in the upper toluene phase while the TMP lysine remains water soluble.
Then remove 200 microliters of toluene using the 20 to 200 microliter multichannel pipette with barrier tips and transfer it into a 96 well quartz plate for reading samples should be clear and free of any aqueous phase. Next, read the absorbance of the samples at 340 nanometers in a Spectra Max three 40 plate reader. Clean the quartz plate by rinsing out the toluene with water and placing the quartz plate in a large glass tray in the fume hood.
Be sure to wear appropriate protection and ensure that the fume hood sash is set to the lower six inch level as the following reaction is highly exothermic and sometimes explosive. Pour 100 milliliters of a seven to three mixture of 70%nitric acid to 95%ethanol over the quartz plate and cover it with the second glass tray after cooling, wash out the nitric acid with water and then 95%ethanol to empirically determine the linear range of the assay. It was performed with various concentrations of cadaver instead of L lysine or LDCI.
The assay was linear to an optical density at 340 nanometers of 0.25 corresponding to approximately 22 M of cadaver. The activity of LDCI alone was determined to be 153.5 plus or minus 18.1 nanomoles of cadaver produced per minute per microgram of LDCI at pH 6.5. The activity of LDCI is unaffected in the presence of 100 micromolar GTP or GDP, but is strongly inhibited greater than tenfold in the presence of 100 micromolar of the stringent response Effectors.
P-P-P-G-P-P and P-P-G-P-P. We've just shown you how to measure the activity of LDCI and we've demonstrated that LDCI is strongly inhibited by P-P-G-P-P in vitro when doing this procedure. It is important to remember to follow the timings accurately at each step of the experiment when doing the toluene extraction.
Make sure to seal the 96 well plate very well. And when taking samples out for absorbance measurements, be sure to remove only the upper toluene phase. So that's it.
Thanks for watching and good luck with your experiments.
