Today, we will demonstrate a method to screen a peptide library, against a recently discovered master receptor, which is Mas-related G protein receptor X2, also known as MRGPRX2 receptor. Mast cells are an integral part of the immune system, and play a crucial role in both innate and adaptive immune responses. Mast cells are activated either by the antigen-bound Fc epsilon R1 receptor, or by the recently discovered X2 receptor.
Activation of surface-bound X2 receptor, has been linked to several immunological, and inflammated diseases and hence, it is important to understand the binding mechanism of this receptor to its ligands. To do so, we have develop a library of small peptide molecules, and have screened them against X2 receptors over-expressed on hexis. In the study, a peptide library was constructed using simple and versatile techniques of Alanine scanning, and amino acid truncations.
Heck293 says, expressing X2 receptors when activated with the peptides and wide-type hexis were used as control. In truncation release upon activation was monitored to study the X2-based activation. Experimentally, Fura-2 AM Calcium Sensitive Dye is excited at 340 and 380 nanometers, while the emission is recorded at 510 nanometers.
Upon calcium binding, the fluorescence intensity at 340 increases, while that of 380 nanometers, decreases. The dye is then deposited at the ratio of the fluorescence intensity at 340, to that of 380 nanometers. The 340 of a 380 ratio, is proportionate to the intracellular calcium, the value of it, can be calculated, by the Grynkiewicz Equation.
The tabbing ratio of two fluorescence intensities, effect of experimental factors like diluting, photobleaching, dye leakage, and scent densities are detected. So, without further delay, let's start the experiment. To identify the ligands of the mast cell MRGPR X2 receptor, generator N-Truncated, C-Truncated, and N+C-Truncated peptide library, by truncating the N-terminal, C-terminal, and N+C-terminal amino acids of pam-12 misproctivity.
Use solid phase peptide synthesis to synthesize the peptides. Modify the end terminal to a set tide group, and C-terminal to amide group. Generate and alanines can peptide library, by replacing the respective amino acids of pam-12 by Alanine, one at a time.
Modify the end terminal to a setide group, and C-terminal to amide group. Prepare culture medium by supplementing high glucose DMEM, with 10%fetal bovine serum, two millimolar L-Glutamine, hundred units per ml of penicillin, and hundred microgram, per ml of a streptomycin. Besides the cells in tee sue culture traited, 375 culture flask at 37 degrees Celsius, 5%CO2, til they are 75 to 80%confluent.
Once 75%confluent, wash the cells and add two to three ml of Proof-C, to detach the cells. Once the cells have detached, collect the cells in Proof-C. Add six to nine ml fresh medium.
Centrifuge the cells at 1620g for three to five minutes. After centrifugation, discard the supernatant to collect the pellet. Resuspend the cells in fresh culture medium.
Dilute the cells, as per the desired concentration. At 200 microliters of the cell suspension with the concentration of 200, 000 cells, pour the ambon in each well to seek 40, 000 cells per well. Guard the cells for 24 hours in the 37 degrees Celsius, 5%CO2 incubator.
Use Fura-2 AM dye for the experiment. Add 50 microliter DMSO in 50 microgram Fura-2 AM, to prepare one millimolar stark solution of Fura-2 AM dye. Add one microliter of one millimolar Fura-2 AM dye per ml of fresh medium to prepare the diluting medium of one micromolar dye concentration.
Remove the 96-well plate from the incubator and discard the medium. Replace the medium with fresh diluting medium. Add 200 microliters of diluting medium in each well.
Incubate the cells for 30-40 minutes in 37 degrees Celsius, 5%CO2 incubator. While the cells are being incubated, set the plate reader. Set the temperature to 37 degrees Celsius.
In settings, select Flex. Set the Read Mode to Fluorescence and Bottom Read. In Wavelengths, set number of wavelengths to two.
Set the Excitation to 340 nanometers and 380 nanometers. Set the Emission to 510 nanometers. Leave the sensitivity to Default.
In Timing, set the interval to 3.9 seconds. Set Run Time to 94 seconds to get 25 Numbers of Reads. Next, select the Assay Plate type.
Next, select the Wells to Read. In Compound Transfer, set transfers to one, and Initial Volume to hundred microliters. Set Pipette Height to hundred microliters, Volume to 50 microliters, and Time Point to 36 seconds to add the compound, and to tend the reading.
Next, select the Compound Source plate type. Leave Triturate to not used. Select the Tips and Pipette Tips Layout.
For Compound and Tip Columns, the compound will be transferred at in column one of the compound plate. Set the Tip Column to one, and Compound Column to one. Leave AutoCalibrate to On.Click OK.After 40 minutes of incubation, remove the medium.
Wash the cells with the XDB buffer. Add hundred microliters of XDB buffer for fluorescence reading. Take the plate for fluorescence reading.
When pritchett has reached 37 degrees Celsius, press the reading chamber, to put the assay plate into the fluorescence plate reader. Press the source to put the compound plate. Prepare the compound plate by adding 200 microliters of respected peptides ionomysin and EGTA to turn X solutions.
Press the tip pratt to put the tip box. Use black tip to avoid tip autofluorescence. Once the plates have been kept, and if you use the settings of the software and press Read.
Determine the Calcium Concentration From the Fluorescence Ratio by the Grynkiewicz Equation. Shown is the fluorescence data of pam good peptide. As can be seen, after peptide addition at 36 seconds, fluorescence at 340 nanometers that is luker increases by that of 380 nanometers record decreases.
Data is represented as the ratio of 340, to that of 380 signal. They show of the signals increases after peptide addition. This figure is the representative data for an activating peptide.
Figure A shows the fluorescence signals for an activating peptide. Representive data corresponds to cram12 Peptide was added after creating a baseline for tendering cycles, as shown by the ado. Figure B shows the ratio of fluorescence emission after excitation at 340 nanometer, to that of fluorescence emission after excitation at 380 nanometers.
This figure is the Representative data for the blank. Figure A shows the fluorescence signals for the blank. HTB was added after diluting a baseline for 10 feeding cycles as shown by the ado.
Figure B shows the ration of fluorescence emission after excitation at 340 nanometers to that of florescence emission after excitation at 380 nanometers. This figure is the Representative data for the standards for dye calibration. Figure A shows that ionomycin was added at the 10th readings, as shown by the ado to get maximum fluorescence in cashem-bound state.
EGPA Triton X hundred was added after 20 readings as shown by the ado who get minimum signal. Figure B shows the ratio of fluorescence emission after excitation at 340 nanometers to that of fluorescence emission after excitation at 380 nanometers. These values are further put in the Grynkiewicz Equation to get the intracellular cache of concentration.
This figure shows the Characterization of a Representative Peptide to Confirm the Sequence and Purity. Figure A shows the vertical mass of the representative peptide sequence WNK WAL was 857.90 dye thining. Which is shown by the N over Zed ratio in Mass Spectroscopy.
Figure B shows a peptide purity of 99%as confirmed by HPLC. This peptide belongs to N+C-truncated peptide library. In conclusion, the method described here is an easy and versatile which can be efficiently and bright for the last scale calcium-based screening.
However, there are several factors which determines the quality of data, and thus, the makeup needs to be optimized for a given cell instrument system. Thank you.
