This procedure begins with overexpression of the recombinant C terminus of the Puric P two X receptor or P two X two, engineered as A GST fusion protein following bacterial lysis. Purified protein is incubated with glutathione seros four B beads, and the concentration is red using the Bradford method. Next proteins from purified rat brain lysate are incubated with the C terminus of P two X two receptors bound to glutathione, seros beads and protein complex is bound to the C terminus of P two X two receptors are isolated via SDS page, gel electrophoresis and excision, and are identified by mass spectrometry.
Hi, I am Harpring from the laboratory of JIT K Department of Physiology at University of California Los Angeles. Hi, I am Sarah Walburn from the laboratory of Tom Vka in the Department of Anesthesiology at the University of California Los Angeles. Today we'll show you the procedure to isolate and purify the CTS of P two X two receptors.
We use these proteins to identify the in from the red brain and in our laboratory we use these proteins to study the functional consequences on P two X two receptors. So let's get started. The cytosolic domain of the P two X two receptor consists of the N and C termini.
The C terminus of the P two X two receptor will be used as bait for the pull down assay, also shown as the sequence of the P two X two receptor C terminus used for this interaction study. To express the full length C terminus of P two X two receptors use conventional molecular biology techniques to integrate the coating sequence for the C terminus of the receptor. Residues 3 53 to 4 72 into a PGE X four NT one plasmid.
The recombinant plasmid is then transformed into e coli strain BBL 21 for expression. The bacteria containing the recombinant plasmid is cultured in LB at 37 degrees Celsius. When the optical density of the culture it's 600 nanometers is between 0.6 and 0.8.
Induce the culture with one millimolar IPTG and incubate for three hours at 37 degrees Celsius. When the three hour incubation is over, remove a one milliliter aliquot from the culture and save for later analysis. Spin down the remainder of the culture at 5, 000 Gs for 15 at four degrees Celsius after centrifugation, discard the supernatant and resuspend the pellet in 20 milliliters of saline tris EDTA buffer transfer the resuspended pellet to a 50 milliliter falcon tube.
The suspension must now be centrifuged at 5, 000 Gs for 15 minutes to collect the semi dry pellet, which is frozen at negative 70 degrees Celsius overnight. On the following day, resus suspend the frozen pellet in 40 milliliters of ice code, lysis, buffer, and put on ice for 30 minutes while the resuspended pellet is sitting on ice. Prepare three milliliters of glutathione seros four B beads.
First wash the beads with 20 milliliters of PBS and 20 milliliters of TPBS. After that, wash the beads with pure PBS and resuspend them in one milliliter PBS. The bacterial suspension in lysis buffer is now freeze thaw the liquid nitrogen.
The frozen sample was incubated on ice to thaw and again purged into liquid nitrogen. This step was repeated three times. Next, sonicate the freeze thaw suspension on ice.
After the sonication, incubate the bacterial suspension with a solution containing 4%tritton X 110 millimolar magnesium sulfate, and two millimolar a TP for 30 minutes on ice. Then spin down the lysate at 20, 000 Gs for 15 minutes at four degrees Celsius and save the supernatant. And before discarding the pellet, take a small sample of the pellet with supernatant to analyze the proteins in the cytosol and membranes.
Incubate the supernatant with the beads for one hour or overnight at four degrees Celsius After their incubation, spin down the beads at 500 Gs for five minutes. Discard most of the supernatant, but save a small sample to represent the fraction of unbound proteins. Wash the pelleted beads five times with 25 milliliters of TPBS while saving a little of each wash for wash controls.
Finally, resuspend the beads and PBS to a 50%weight to volume slurry and stored at four degrees Celsius. The protein concentration can be estimated using the Bradford assay as per the manufacturer's instructions and is expected to be 500 micrograms per milliliter. To identify the binding partners of P two X two CT GST from whole brain Lysates, a pull down assay is performed using CT GST immobilized on the glutathione coated beads as bait as a control.
Use GST alone bound to beads as bait here forth referred to as the GST Knoll Rat Brainin lysate. For the GST Fusion Protein Binding assay was prepared using a standard protocol and lysis buffer and protein concentration was estimated as 15 milligrams per milliliter by the Bradford assay. Before beginning the assay.
Pre-clear the thaw brain lysate by incubating it with 30 microliter GST beads for one hour at four degrees Celsius after the lysate has been incubated with the GST beads. Spin down the beads at 1000 Gs for 10 minutes at four degrees Celsius. Save the supernatant and keep a small volume of it as a control.
Now calculate the volume of lysate that should contain 100 micrograms of the pre-cleared solubilized protein and combine this volume of lysate with 10 micrograms of GST bound beads and 10 micrograms P two X two CT GST bound beads. Make up the volume to 200 microliters using lysis buffer. Incubate the lysate with beads overnight in a rotator at four degrees Celsius.
The next day, spin down the beads at 1000 Gs for five minutes and discard the supernatant. Wash the beads three times with the one milliliter lysis buffer. Then add modified 50 microliters lely buffers to the beads and boil the beads in this buffer for five minutes.
Spin down the boiled samples at 5, 000 GS for five minutes and run the sate in doubt on a 10%SDS page on the gel. Compare the P two X two CT associated proteins with those recovered using GST Nola's bait. The final critical step of the analysis is to remove the separated proteins from the gel for identification by mass spectrometry from this point forward.
An absence of dust throughout the process is critical to reduce keratin contamination. Begin by cutting the bands from the gel with a clean razor blade. Then dice the bands into three smaller sections and put each band in a labeled micro centrifuge tube corresponding regions of the gel in the GST.
No pull down lane must also be excised in the same manner without regard to band staining. To ensure the presence of proteins below the level of staining sensitivity are not missed, mix the gel pieces with 200 microliters of 50 millimolar ammonium bicarbonate and 50%volume by volume. Acetonitrile then vortex the tubes for 15 minutes at room temperature.
After vortexing, remove the wash solution and repeat. Remove the final wash solution and dehydrate the gel pieces by adding 200 microliters aceto nitrile. The gel pieces should shrink and become opaque in color.
Within two minutes, remove the acetyl nitrile and dry the gel pieces. Using a speed vac for 10 minutes, the dry gel pieces are next incubated in a freshly prepared 10 millimolar DTT 10 milli molar TEP solution. The volume of the solution, usually about 30 microliters must be sufficient to immerse the gel pieces.
Allow the gel pieces to be reduced in the solution for 30 minutes in a 56 degree Celsius heat block. Next, remove the DTT TEP solution and replace it with a wash solution of 100 microliter 500 millimolar ammonium bicarbonate in 50%Acetonitrile vortex for 10 minutes after vortexing aspirate off the ammonium bicarbonate wash and replace it with 200 microliters of Acetonitrile to dehydrate the gel pieces. The gel pieces should shrink and become opaque in color within two minutes.
Next to Alkylate, any free sulf hydros, replace the acetyl nitrile with 100 microliters of freshly prepared 100 millimolar I OTO Acetamide solution. Incubate this mixture for 25 minutes at room temperature in the dark. After the incubation, remove the IDO Acetamide solution and wash the gel pieces with 200 microliters of 50 millimolar ammonium bicarbonate and 50%Acetonitrile by vortexing for five minutes.
This wash step is repeated once. Remove the last wash solution and incubate the gel pieces in 200 microliters acetyl nitrile for 10 minutes. Remove the acetyl nitrile and dry the samples and the speed vac.
At this step. The gel pieces are ready for trypsin digestion. Apply 30 microliters of trypsin solution at a working concentration of approximately 20 nanograms per microliter and incubate on ice for 10 minutes.
The gel pieces should become well swollen. Remove the excess trypsin solution and immerse the rehydrated gel particles in 30 microliters of 50 millimolar ammonium bicarbonate. Allow the digestion to proceed for 12 to 16 hours at 37 degrees Celsius after the long digestion at five microliters of 5%formic acid to deactivate the trypsin and to arrest digestion.
Then vortex the tubes for 15 minutes after vortexing centrifuge the tubes briefly to bring the liquid to the bottom of the tube and transfer this solution containing the digested peptides to a clean labeled half mill tube. Next, cover the gel pieces with about 30 microliters of extraction solution containing 0.1%formic acid in 50%acetyl NI trial and vortex for 10 to 15 minutes. Add this extraction solution to the new half mill tube.
Repeat this step. Transferring the extraction solution to the new half mill tube, the half mill tube containing the overnight digestion solution and the two extraction solutions is reduced to approximately 30 microliters in the speed vac. This peptide solution is now ready for analysis by L-C-M-S-M-S.
Separate the proteins by reverse phase nano lc, a C 18 reverse phase column, acquire spectra and data dependent mode with the orbitrap used for MS scans and LTQ for MS to MS scans. Identify proteins by searching the data against the rat IPI database using the sequester algorithm integrated into the Bioworks software package, and then manually inspect the spectra only proteins that are present in each of the four sample replicates and are absent during GST. No pull downs should be considered for further analysis.
Here is the Cipro stain gel showing a spectrum of putative proteins that interact with the C terminus of P two X two receptors. Fused with GST indicated by the blue box control lanes for GST alone indicated by the yellow box and glutathione. Sphero speeds alone are also shown.
The arrows indicate examples of unique bands that were excised for further analysis by mass spectrometry. We have just shown you how to express and purify the CTOs of P two X two receptors. We have also shown you that how to perform the pull down acid and identify the protein interactors.
It's important to remember when doing this procedure that you wear gloves and a hairnet to reduce the keratin contamination in your samples. So that's it. Thanks for watching and good luck for your experiments.
