The overall goal of this procedure is to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid. This method can help answer key questions in the chemical biology field such as in protein modification, engineering and conjugation. The main advantage of this technique is that your target probe or broth can be site-specifically introduced into proteins or antibodies by simple mixing.
After constructing the plasmids, add one microliter of each to 20 microliters of E.Coli strain DH10-beta at a concentration of two times 10 to the eight cells per microliter to a 1.5 milliliter centrifuge tube. Use a pipette to gently mix the solution. Then place 20 microliters of the mixed solution into a clean cuvette.
Set the electroporation machine to 25 microfarads and 2.5 kilovolts. Then insert the cuvette into the slide of the shocking chamber. Push the slide into the chamber until the cuvette makes firm contact with the chamber electrodes.
Press the pulse button once until the electroporation machine beeps. After this, add one milliliter SOC medium to the cuvette quickly. Then transfer the mixture to a test tube.
Incubate with shaking at 37 degrees Celsius for one hour. After incubation is complete, spread the transformed E.Coli cells onto lysogeny broth agar plates containing appropriate antibiotics. Incubate the plates at 37 degrees Celsius for 12 hours.
After the plate incubation is complete, inoculate a single transformed colony in five milliliters of LB medium containing antibiotics. Incubate with shaking for 12 hours at 37 degrees Celsius. Then transfer the primary culture into 200 milliliters of LB medium with 100 micrograms per milliliter ampicillin and one millimolar AF.Incubate with shaking at 37 degrees Celsius for at least three hours.
Add two milliliters of 1.6 molar arabinose when the culture reaches an optical density of 0.8 at 550 nanometers. Then incubate with shaking at 30 degrees Celsius for 12 hours. When the incubation is complete, harvest the cells by centrifugation at 11, 000 times g for five minutes.
Discard the supernatant and freeze the pellet at negative 20 degrees Celsius. When ready to lyse the cells, re-suspend the pellet in 20 milliliters of paraplasmic lysis buffer in a fresh centrifuge tube. Incubate the mixture at 37 degrees Celsius for one hour.
Next, centrifuge the cell lysate at 18, 000 times g for 15 minutes at four degrees Celsius. Transfer the supernatant to a fresh tube and discard the pellet. Then add 400 microliters of an Ni-NTA resin suspension.
Using a rotator, gently agitate the tube for one hour at four degrees Celsius. After mixing is complete, pour the suspension into a polypropylene column. Wash the resin three times with five milliliters of wash buffer.
Elute the target antibody with 300 microliters of elution buffer. After this, determine the concentration of the mutant protein as outlined in the text protocol. Begin by adding 10 microliters of a 10 micromolar solution of HerFab-L177AF in phosphate buffer containing 10 millimolar sodium phosphate dibasic and 100 millimolar sodium chloride to a fresh centrifuge tube.
Then add 20 microliters of a 200 micromolar solution of Cy 5.5 azadibenzocyclooctyne in water. After this, cover the reaction vessel with aluminum foil. Allow the stain-promoted cyclo-edition reaction to proceed for six hours at 37 degrees Celsius.
Once the reaction is complete, add 30 microliters of sample and 450 microliters of phosphate buffer to wash free Cy 5.5 dye in a centrifugal filter spin column. Centrifuge the spin column at 14, 000 times g for 15 minutes at four degrees Celsius. Discard the flow-through and then transfer the purified sample to a 1.5 milliliter microcentrifuge tube.
Store the purified labeled HerFab at four degrees Celsius unless ready for analysis. Begin by adding 13 microliters of 7.8 micromolar purified HerFab to a fresh tube. Then add five microliters of LDS protein sample buffer.
Incubate the mixture at 95 degrees Celsius for 10 minutes. After incubation, attach the four to 12%Bis-Tris SDS-PAGE gel cassette to the electrophoresis cell. Add running buffer.
Next, load the conjugated protein samples in the pre-stained molecular weight maker. Perform gel electrophoresis for 35 minutes at 200 volts. After the electrophoresis is complete, transfer the gel to a gel documentation and analysis system.
Scan for Cy 5.5 and then stain the gel with a commercial protein stain. In this study, the antibody fragment Her-Fab is site-specifically conjugated with a This is accomplished by incorporating an azide-containing amino acid into the fragment and then reacting the mutant antibody fragment with a strained cyclooctyne. SDS-PAGE analysis is performed for mutant proteins expressed both with and without the azide-containing amino acid.
As can be seen, the full-length antibody fragment was obtained only in the presence of the amino acid. The antibody containing the amino acid is evaluated for conjugation via reaction with a Cy 5.5 linked azadibenzocyclooctyne derivative Cy 5.5 and analyzed by SDS-PAGE. The fluorescence images show conjugation of the mutant antibody fragment with Cy 5.5 while no conjugation was observed in the control.
Once properly expressed and purify your protein with acetylphenylalanine, the protein can be efficiently labeled with a probe with the strain Because the labeling reaction requires no additional reagent, you can introduce the probe into your target protein by simply mixing the probe with your protein. After watching this video, you should have a good understanding of how to site-specifically introduce chemical probe into an antibody fragment by genetically incorporating an azide-containing amino acid.
