The overall goal of this method is to label antibodies with fluorescent dies from purified samples or directly from cell medium using magnetic protein A or G beads. This method can be used to optimize the labeling chemistry of even small amount of antibodies within the cell medium. Leading to better downstream application of the antibodies.
The main advantages of this technique are that the antibodies do not require purification, amine or thiol chemistry can be used, and the method can be automated or manual. Begin by uniformly re-suspending the beads with gentle shaking. Then, add 50 microliters of beads to a 1.5 milliliter microsentrifuge tube.
Then, place the tube in a magnetic stand. After 10 seconds, carefully replace the storage buffer with 250 microliters of antibody-binding buffer and mix the beads well. After another 10 seconds, carefully replace the binding buffer with one milliliter of the antibody of interest.
And mix the sample for 60 minutes at room temperature. At the end of the incubation, return the tubes to the magnet and remove the supernatant when the beads have moved to the sides of the tube. Then, rinse the beads and antibody mixture by two 10 second washes with 250 microliters of antibody-binding wash buffer per wash.
To label the antibodies using amine chemistry, after the second wash, replace the wash buffer with 100 microliters of amine conjugation buffer and add 2.5 microliters of freshly prepared amine-reactive dye per 100 micrograms of anitbody. Vortex the sample for 60 minutes at room temperature to keep the beads in suspension during the labeling. Then, return the tube to the magnet for 10 seconds and remove the supernatant.
Wash the beads two times with antibody-binding wash buffer, as just demonstrated. Then, replace the wash buffer with 50 microliters of elution buffer and mix the beads for five minutes. Return the tube to the magnet for 10 seconds and transfer the eluted antibody to a new microcentrifuge tube containing five microliters of neutralization buffer.
Elute the antibody one more time, as just demonstrated, pulling the eluted samples. Then, measure the absorbance of the antibody-dye conjugate at 280 nanometers and at the lambda max for the dye. To label the antibodies using thiol chemistry, after the second wash, replace the wash buffer with 250 microliters of thiol conjugation buffer and mix the beads with gentle pipetting.
After 10 seconds, wash the beads two times with antibody-binding wash buffer. Then, replace the wash buffer with 100 microliters of thiol conjugation buffer and add DTT to a final concentration of 2.5 millimolar. Mix the beads for another 60 minutes.
Then, return the beads to the magnet for 10 seconds and discard the buffer. Next, add 250 microliters of thiol-conjugation buffer to the beads with gentle mixing. After 10 seconds, wash the beads two times with antibody-binding wash buffer, replacing the second wash with 100 microliters of fresh thiol-conjugation buffer.
Now, add 2.5 microliters of freshly prepared thiol-reactive dye per 100 micrograms of antibody and mix the beads for 60 minutes at room temperature. At the end of the incubation, return the tube to the magnet for 10 seconds. And replace the supernatant with 250 microliters of fresh thiol-conjugation buffer with gentle mixing.
After 10 seconds, wash the beads two times. Then, replace the wash buffer with 50 microliters of elution buffer and elute the conjugated antibody two times, as just demonstrated. Then, measure the absorbance of the antibody-dye conjugate at 280 nanometers and at the lambda max for the dye.
The strong affinity between the antibodies and Protein G, the robust affinity of the magnet for the beads, and the optimization of the method prior to the bead purification together result in a nominal loss of antibody during the labeling reaction, as evidenced by the efficient recovery of three different mouse antibody isotypes after fluorescent dilabeling compared to simple purification. This recovery is also evident in Coomassie gels, where the low non-specific binding properties of the magnetic G Protein beads result in the presence of highly purified fluorescence-dilabeled heavy and light chains. On bead labeling with magnetic Protein A beads, it is also compatible with multiple fluorescent dyes, resulting in high recovery and good dye-to-antibody ratios.
Once mastered, this technique can be completed in two to three hours, if it is performed properly. Allowing multiple samples to be handled manually or an automated platform for improved throughput. While attempting this procedure, it is important to remember to keep the magnetic beads in suspension at all times.
Choose the appropriate volume of magnetic beads for the sample. And optimize the amount of fluorescent dye for the antibody of interest. Following this procedure, the labeled antibodies should be tested to determine functionality using relevant biological assays, like ELISA, facts, or cell-based assays.
After watching this video, you should have a good understanding of how to label purified and unpurified antibodies with fluorescent tags or other small molecules, like biotin or cytotoxic drugs, using magnetic Protein A or G beads.
