The overall goal of this procedure is to measure the tRNA as methyltransferase activity of TBA histolytica DNM T two Emmett Enzyme. This is accomplished by first preparing an adequate TRNA ASPA substrate. As a second step, the methyltransferase DNM T two is purified as an active recombinant enzyme following preparation of the active enzyme, the DN MT two enzyme and the TRNA ASPER mixed.
The reaction is then initiated by adding the radio labeled s Aden Methionine cofactor. The final step of the procedure is to stop the reaction and to separate labeled TRNA ASP from unincorporated cofactor. Ultimately, results can be obtained that show the incorporation of the labeled methyl group into the TRNA ASP substrate.
Hi, I am Mark Helm from the Institute of Pharmacy and Biochemistry at the Johannes Gutenberg University of Mikes. I'm Benjamin Mann from the Mark. Hi.I am re from the Molecular Parasitology Lab at the Microbiology Department of the Teon, Haifa Israel.
Hi, I am a Tovi Froms Andre lab. Okay, so today I'm going to show you procedures to observe the TNA asthma transfer activities of the anti DMT two ollo, EH met. We use this procedure often in our lab to study the effect of environmental stresses including glucose starvation on the ANTIER lya, DNA ANDT NA methylation status.
So let's get started. This part of the procedure describes an in vitro runoff transcription, which can be used for the synthesis of any TRNA for this purpose. A DNA template was designed with the complementary sequences of homosapiens TRNA ASP and hammerhead rib zyme.
Complementary to the nine five prime bases of the TRNA at the five prime end include the T seven promoter sequence containing a six nucleotide sequence enabling efficient transcription. After transcription, the hammerhead rib zyme will cleave itself off the transcript, leaving the rib zyme and the full length TRNA. The TRNA can then be separated from the rib zyme and UNC cleaved products by urea page.
A forward primer that is universally applicable for different tRNAs was designed for this experiment. The reverse primer has a similar melting temperature compared to that of the complete forward primer. To ensure binding of both primers to obtain efficient amplification for amplification of the template DNA.
Begin by using a standard PCR reaction in a final volume of 50 microliters. Perform the preparation on ICE using the final concentrations found in the written protocol. Once the reactions are prepared, perform the PCR program.
Use a low eeling temperature during the initial 10 cycles to account for the partial binding of the forward primer to the DNA template. To reduce unspecific binding. Increase the annealing temperature for a further 25 amplification cycles following amplification.
Test the success of template amplification using a 1.3%AGROS gel prestained with one x gel red solution. Next, prepare five x concentrated T seven premix. Then prepare the transcription reactions in 400 microliter reaction volumes using 80 microliters of TT seven premix 80 microliters of PCR product.
80 microliters of NTP mix 145 microliters of milli Q water and 15 microliters of T seven RNA polymerase to start the reaction, carry out the transcription reaction for four hours. At 37 degrees Celsius, a white precipitate of pyrophosphate indicates successful transcription To purify the product first, spin down the pyrophosphate for one minute. At 10, 000 RPM, remove the SUPERNAT and add one volume of foram IDE loading dye.
Run the sample on a 12%urea poly acrylamide gel at 20 watts for two hours. Afterward, stain the gel with three x gel. Red solution supplemented with 0.1 molar sodium chloride for 20 minutes.
Identify and mark the TRNA band upon UV excitation. Excise the TRNA bands with a scalpel and put them into 1.5 milliliter eend cups. Then perform sodium acetate treatment as described in the written protocol.
The next day, remove the sodium acetate supernatant from the samples. Filter the supernatant with nano tubes, spinning them for 90 seconds at 8, 500 RPM to remove page debris, precipitate the alluded tRNAs by adding 2.5 volumes of pure ethanol stored at minus 80 degrees Celsius. Then store the samples overnight at minus 20 degrees Celsius following overnight incubation centrifuge the samples for 2.5 hours at four degrees Celsius and 15, 500 RPM.
Remove the supra natin and dry the pellets in a speed back. Finally, resuspend the pellets in Milli Q water. Determine the concentrations using a NanoDrop ND 1000.
The yield of one 400 microliter transcription should be about 80 to 100 micrograms. Prepare BL 21 e coli expressing the e histolytica EMIC containing PJX four NT one vector as described in the written protocol. Inoculate 500 milliliters of two x yeast extract tritone medium with 100 micrograms per milliliter ampicillin from an overnight culture of the bacteria.
Incubate the cells in an orbital shaker at 37 degrees Celsius until the OD 600 of the culture reaches 0.8. Induce the expression of the DNM T two recombinant protein by adding IPTG to a final concentration of 0.5 millimolar. Continue to incubate the culture in an orbital shaker for 16 hours at 24 degrees Celsius.
This is the optimal time and temperature found to avoid the formation of inclusion bodies following incubation. Harvest the cells by centrifugation at four degrees Celsius and 6, 000 RPM in 200 milliliter Nalgene tubes for 15 minutes. Discard the S supernatant and freeze the pellet at minus 70 degrees Celsius for at least one hour.
Then lice the cells by Resus, suspending the pellet in 30 milliliters of lysis buffer, keeping the samples on ice. Sonicate the sample in an ice water bath. Remove any bacterial DNA that may be attached to Emmett with Ben Nase.
Nuclease treatment for 30 minutes on ice. Complete lysis by addition of 300 microliters of bug buster protein extraction reagent followed by an incubation of 30 minutes at four degrees Celsius with gentle agitation on an orbital shaker. Purify the recombinant GST emett protein under native conditions on a glutathione aeros resin.
According to the manufacturer's instructions, elute the recombinant emett protein from the glutathione aero beads by incubating the beads with five milliliters of glutathione elution buffer for 12 hours at four degrees Celsius with gentle rotation. Following incubation and centrifugation of the sample. Collect the snat load five milliliters of SUP natin on the top of a millipore MicroCon YM 30 centrifugal filter device and centrifuge at 8, 000 RPM for 20 minutes at four degrees Celsius.
Dilute the concentrated protein in five milliliters of storage buffer and reload it onto the filter device. Repeat the same procedure at least three times to ensure complete replacement of the elucian buffer by the storage buffer. Finally dilute the concentrated protein in approximately 300 microliters of storage buffer.
Measure the concentration of the recombinant protein by the Bradford Method. Final concentration of the enzyme should not be less than three micrograms per microliter for the methylation assay. To begin the in vitro TRNA methylation assay prepare a pipetting scheme for a 40 microliter final volume methylation assay.
This scheme should include a negative control that includes Emmett only a negative control that includes TRNA only and a positive control with human D NMT two, which has 10 times more activity than Emmett. This scheme should also comprise a standard reaction that includes the enzyme, the substrate, and the co-factor, as well as the total value of the adamant on the filter. This value is used for the calculation of the percentage of methyl group incorporated To begin the assay dilute TRNA with DEPC treated water to a final concentration of five micromolar in a 1.5 milliliter eend orph tube in a separate tube combined four microliters of DTT and eight microliters of five x methylation buffer.
Incubate the TRNA solution at 85 degrees Celsius for two minutes and immediately add it to the DTT five X MB solution. Allow the mixture to cool down at room temperature for 15 minutes to let the TRNA fold correctly. Next, prepare the adamant mix by mixing 50 micromoles of labeled OMI with 150 micromoles of unlabeled omi.
Add four microliters of the mix to the TRNA solution and incubate for two minutes at 37 degrees Celsius, add the TRNA methyl transferase Emmett to the solution containing the TRNA substrate and the labeled unlabeled Adamant cofactor to a final concentration between one and 10 micromolar. Incubate the mixture for three hours at 37 degrees Celsius throughout the incubation. Mix the tubes regularly by gently flipping them.
Take an eight microliters sample every 20 minutes over a period of three hours to build a kinetic curve of the enzymatic reaction. Immediately spot each sample on the center of a watman filter with the exception of the Adam Met sample. Soak the filter in a 5%TCA solution while agitating on ice for 10 minutes.
Repeat the 5%TCA wash twice for each sample. Wash the filter with 100%ethanol on ice for 10 minutes. Air dry the filter.
Place each dried filter in a tube that was previously filled with three milliliters of scintillation liquid. Determine the radioactivity incorporate into the TRNA by using a scintillation counter following amplification. The success of template amplification was tested using a 1.3%aeros gel prestained with one x gel red solution.
PCR reactions on two different templates are shown alongside a 100 base pair. Plus ladder controls include a template only lane and reactions in which the reverse primer was omitted. If the expected yield of the transcription reaction loaded onto the urea poly acrylamide gel is above 50 micrograms.
Bands can be seen by UV shadowing A page. Separation of two transcription mixtures is shown. Bands of precursor transcripts full length TRNA and hammerhead ribo design become visible as shadows since they prevent UV light from reaching the fluorescent TLC plate Total, TRNA from e coli is used as a size marker here.
Examples of values read by the scintillation counter are shown based on the scheme described in the protocol. These results include a negative control that includes mbit only a negative control that includes TRNA only and a positive control with human DN MT two, which has 10 times more activity than Emmett. The assay also included a standard reaction that includes the enzyme, the substrate, and the co-factor, as well as the total value of the adamant on the filter calculation of one unit of Emmett Methyltransferase activity is expressed as the incorporation of one PICA mole of ate per hour per nano mole of protein.
We have just shown you over TRNA asthma transferase activities of the Anica D two met When doing this procedure. It's important to avoid the degradation of the TNA as substrate and to always keep the DN T two enzyme on ice. Indeed, a freshly prepared DTT solution is essential.
So that's it. Thank you for watching And good luck with your experiments minutes.
