The overall goal of this procedure is to identify abers from a random library, which specifically bind to a target protein in this case, S 100 B protein. This is accomplished by first digesting the library with restriction endonuclease to liberate random sequences from flanking fixed primer sequences. The second step of the procedure is to bind the random library sequences to purified S 100 B protein and recover them.
The third step of the procedure is to reunite bound sequences with fixed primer sequences by hybridization and ligation. The final step of the procedure is to transcribe the ligated products to allow R-T-P-C-R re amplification of the bound pool of candidate abers, followed by additional rounds of this selection procedure. Ultimately, results can be obtained that shows specific binding of selected abers to the S 100 B protein.
For example, here, sandwich binding assays are performed using microarrays and fluorescently labeled second abers assays are also performed using functionalized nanowires and second abers coupled to 15 nanometer gold nanoparticles. Today we will show a procedure for selecting optimal, which bind to S hundred B protein from random library of D sequences. We use this procedure in our laboratory for a variety of purposes, including developing a sensor platform for the early detection of cancer.
So let's get started. To begin this procedure, generate a primer free or PF DNA library as previously described. Cleavage of the double stranded DNA library followed by gel purification with a 10%denaturing poly acrylamide gel will result in a 32 nucleotide fragment of five prime phosphate in 30 cc, and a 30 nucleotide fragment of five prime phosphate in 30, designated the 32 plus and 30 plus fragment respectively.
The 32 plus fragment contains the 30 nucleotide random domain with the CC minimal flanking sequence at the three prime end, whereas the 30 plus fragment consists only of the 30 nucleotide random domain sequence. To prepare the PF library DNA fragments for aptr selection, resus suspend each of the 32 plus and 30 plus fragments in 40 microliters of 20 milli tris, HCL pH 7.4. Then heat the samples for three minutes at 85 degrees Celsius following incubation.
Cool, the samples down for three minutes in a 37 degree Celsius water bath incubator, next at 760 microliters of selection, buffer and incubate for three minutes at 37 degrees Celsius. After three minutes, keep the sample at room temperature for 10 minutes. Following incubation, pass the solution through a column containing nickel NTA agro beads pre-washed with selection buffer.
To remove nonspecific DNA, collect the PF library DNA fragments that do not bind to the column and set aside for later. To prepare the S 100 B bound beads first spin 400 microliters of nickel NDA beads for three seconds in a micro centrifuge and discard the supernatant. Then wash the beads with 400 microliters of binding buffer by gently pipetting five times.
Then spin down the beads and discard the supernatant. Repeat this. Wash twice.
Next poor previously purified histidine tagged protein over the beads. Here, human S 100 calcium binding. Protein B is used gently pipet the solution up and down five times every three minutes for a total of 15 minutes.
After 15 minutes, spin on the beads and discard the supernatant. Finally, wash the bead bound S 100 B with 400 microliters of selection buffer by gently by petting up and down three times. Again, spin the beads down and discard the supernatant.
Repeat this step twice for a total of three final washes to select for S 100 B bound abers. Retrieve the 32 plus and 30 plus fragments that were previously put aside. Add the fragments to the bead bound S 100 B.Then incubate the sample for 15 minutes.
Mixing every three minutes by gently pipetting up and down after 15 minutes, briefly spin down the beads, discard the supernatant, then wash the S 100 BDNA complex with 800 microliters of selection buffer by gently pipetting. Then spin the beads down and discard the supernatant. Repeat this washing step twice.
To recover the S 100, be selected abers at 200 microliters of 20 millimolar tris, HCL pH 7.4 to the beads. Then heat the slurry for three minutes at 85 degrees Celsius. Following incubation.
Vortex the beads for one minute and spin them down. Then transfer the supernatant to a fresh tube. Repeat this step once and combine the supernatants.
Finally, purify the selected DNA fragments. Extract the PCR products twice with an equivalent volume of phenyl chloroform, isopropyl alcohol. Precipitate them with 250 millimolar sodium chloride and 2.5 times the volume of 100%Ethanol at minus 70 degrees Celsius for 15 minutes.
Spin down at 21, 000 GS at four degrees Celsius for 15 minutes. Then discard the supernatant. Rinse the pellet with 75%ethanol efficiently.
Spin down at 21, 000 GS at four degrees Celsius for five minutes and discard the supernatant. Dry the pellet in a speed vacuum for three minutes After 10 rounds of selection. Clone the resulting PCR amplified DNA fragments into the PCR 2.1 topo vector obtained from a vitrogen core.
Then sequence between 40 and 50 single colonies for each of the selected N 30 cc and N 30 fragments with the M 13 forward primer. Align the selected sequences using infomax vector NTI software. Then determine the consensus sequences based on the alignments.
Once consensus sequences have been identified, purchase consensus abers as described in the written protocol. Suspend the first aptamer synthesized with a five prime Amin C six moiety in printing buffer to a final concentration of 15 micromolar. Then spot the aptamer solution onto code link activated slides using an apogen discoveries microgrid array.
Following code link recommended protocols. Print each slide with 12 arrays. Use a two by eight format.
Microarray hybridization cassette for hybridization. Dilute the S 100 B protein to the appropriate concentration in 70 microliters of PBS. Then apply the solution to the wells of the microarray cassette.
Seal the microarray cassette using nuclease free adhesive ceiling foil to prevent evaporation. Allow the protein to bind to the abers printed on the code link. Slide for one hour at room temperature following incubation individually.
Wash the wells of the cassette three times with PBS five millimolar magnesium chloride or PBSM. Next, dilute the fluorescently labeled second abers to 0.125 micromolar in 70 microliters of PBSM. Heat the mixture for three minutes at 85 degrees Celsius.
Then allow the labeled ABERS to cool on ice. For three minutes, apply the abers to the wells of the microarray cassette. Again, seal the cassette with the adhesive foil and incubate them for one hour at room temperature.
Again, individually. Wash the wells three times with PBSM following the wash. Disassemble the cassette and rinse the whole slide with PBSM.
Then thoroughly dry the slide by centrifugation. Finally, scan the slide using a scanning Packard Biosciences scan array. 4, 000 XL binding was determined for gamma P 32 A TP five prime end labeled aptamers to S 100 B protein and plotted against concentration for KD determination.
Representative kds after approximately seven rounds of selection are typically in the 10 to the negative seven molar range. Five prime aiming dert first aptamers were coupled to code link microarrays purified S 100 B protein was bound to the first optimer and LOR 5 46 labeled second. Optimer was bound to the first optimer S 100 B complexes.
Fluorescence was quantitated using a scan array scanner. The middle row shows sandwich binding wither's characterized by additive binding in KD assays in the rows above and below. There is no sandwich binding for abers five prime thiol derivatized first abers were coupled to gold nanowires using standard thile chemistry.
Second abers were coupled to 50 NMO or gold nps in the same manner. Purified S 100 B protein was then bound first to the deriv nanowires second aptamer 50 nano molar gold mps were subsequently bound to the first aptamer nanowire S 100 B complexes. Bound sandwich complexes were visualized using F FE SEM.Here.
A negative control can be seen for a binding assay performed with the HTRA one protein in place of the S 100 B protein. Okay, so one of the most important things about the procedure is that we're using a primer free approach where we don't have fixed sequences which can cause false positives and false negatives based on the the, the sequences themselves overwhelming the random region. When doing this procedure, it's important to make sure the DNA template is destroyed prior to each reverse transcription reaction, and to be careful to isolate the specific R-T-P-C-R product after each re amplification.
So that's it. Thank you for watching and good luck with your experiments.
