The overall goal of this procedure is to produce and purify BDNF oviduct in an optimized protocol involving transfection of HEK293 cells and purification in a chromatography column. The BDNF oviduct is then mono-biotinylated by an in vitro reaction for posterior labeling and detection by fluorescence microscopy. The main advantage of this procedure is that it allows the production, purification, and mono-biotinylation of BDNF in an optimized manner.
The gestation parameters have been optimized for maximum BDNF production using a cost-effective transfection reagent and the purification process is performed in a chromatography setup that facilitates manipulation and allows to escalate the procedure. Begin by diluting 60 micrograms of polyethylenimine in 500 microliters of unsupplemented DMEM and 20 micrograms of BDNF oviduct plasmid in 500 microliters of unsupplemented DMEM per 15 centimeter dish to be transfected. Incubate for five minutes at room temperature and then carefully pipette the DNA solution into a polyethylenimine tube, mixing once by up/down motion.
Incubate for 25 minutes at room temperature and then drip one milliliter of the PEI/DNA mixture throughout each 15 centimeter dish. Incubate the cells with the PEI/DNA mixture for three hours and then change the medium to incubation buffer. 48 hours after the transfection of the cells, collect the medium from all the dishes.
Then add the BDNF medium components to the HEK293 supernatant including sodium chloride, phosphate buffered components, imidazole, and protease inhibitors. Incubate in ice for 15 minutes. Then aliquot the medium into centrifuge tubes.
Centrifuge for 45 minutes at 10, 000 g in a four degree Celsius gold centrifuge. This step allows the elimination of cell debris and dead cells suspended in the media. Then collect the supernatants.
Add BSA at a final concentration of 0.1%and store at minus 20 degrees Celsius for up to two months. To purify the BDNF, begin by thawing the media in a 37 degree Celsius thermoregulated bath. Then aliquot the media into centrifuge tubes.
Centrifuge for one hour at 3, 500 g in a four degree Celsius gold centrifuge. This step allows the elimination of remaining debris to ensure adequate flow through the chromatography column. Then use the protein concentrators to reduce the media from 500 milliliters to 100 milliliters.
Follow the manufacturer's recommended centrifugation parameters for optimal concentration. Then add 500 microliters of nickel-NTA agarose beads to the concentrated media and incubate overnight in a rocker at four degrees Celsius. The next day, assemble the chromatography apparatus and pour the media into it.
Let it rest for five minutes and then open the two-way stop cock to let the medium flow through. Wash with five milliliters of wash buffer for five minutes making sure to resuspend the beads in the column. Repeat three times.
Finally, add one milliliter of elution buffer to the column making sure to resuspend the beads. Incubate for 15 minutes and then collect the eluent in a microcentrifuge tube. Repeat this step three times for complete elution of BDNF.
To biotinylate the BDNF, take an aliquot containing 800 nanograms of the protein and add the biotinylation buffer reagent and then some BirA-GST in a one-to-one molar relation to BDNF. Incubate in a hybridization oven at 30 degrees Celsius for 60 minutes, mixing the contents of the tube every 15 minutes by inversion. Then add the same volume of ATP and BirA-GST as in the first step and incubate for 60 minutes, mixing the contents of the tube every 15 minutes.
The biotinylated BDNF can then be left in ice for immediate biotinylation verification or stored at minus 80 degrees Celsius for posterior analysis. To verify the biotinylation of the BDNF oviduct, begin by blocking 30 microliters of streptavidin magnetic beads per BDNF sample in a blocking buffer. Incubate at room temperature for one hour and then precipitate the magnetic beads in a magnetic rack separator and then discard the blocking buffer.
Add 50 microliters of fresh blocking buffer and the BDNF sample to the beads, making sure to resuspend them completely. Homogenize the contents of the tube and incubate at four degrees Celsius for one hour in a microcentrifuge tube rotator. Prepare the sample for western blotting as described in the text to assess the efficiency of the biotinylation reaction.
To conjugate the BDNF oviduct to the quantum dots, begin by adding the necessary volume of quantum dots to achieve a one-to-one molar relation to BDNF and then dilute to a final volume of 20 microliters. Homogenize the contents of the tube and then wrap it in aluminum foil to protect it from the light. Incubate at room temperature for 30 minute in a rocker.
Then dilute the BDNF oviduct to the desired final concentration and add it to the axonal compartment of a microfluidic chamber, incubating for 210 minutes for posterior live or fixed cell imaging. The use of a chromatography column-based protocol allows the processing of significant volumes of HEK293 conditioned media. In this experiment, 500 milliliters of conditioned media were processed to purify BDNF oviduct.
Four consecutive elutions lasting 15 minutes each yielded decreasing concentrations of BDNF oviduct ranging from six to 28 nanograms per microliter. The total yield amounted to approximately 60 micrograms of BDNF oviduct. This purified BDNF was then biotinylated by an in vitro reaction mediated by BirA-GST.
The sample was homogeneously biotinylated as demonstrated by the lack of non-biotinylated BDNF oviduct in the supernatant of the magnetic bead cleared buffer. These results show that the proposed protocol allows for the purification of significant amounts of BDNF oviduct and homogenous in vitro mono-biotinylation. Then the biological activity of the mono-biotinylated BDNF was evaluated using two different experimental approaches.
First, cortical neurons seeded in 60 millimeter plates were stimulated with 50 nanograms per milliliter of mono-biotinylated BDNF for 30 minutes and then proteins were prepared for western blot analysis. The biological activity of the mono-biotinylated BDNF was quantified by detecting phospho-TrkB and phospho-ERK. Binding of BDNF to TrkB triggers the auto-phosphorylation of the receptor and the activation of several downstream kinases including ERK.
A negative control condition which wasn't stimulated with BDNF and a positive control condition which was stimulated with commercially available BDNF were included for the analysis of BDNF oviduct biological activity. The bands were both phosphorylated proteins, had a similar intensity in neurons treated with commercial BDNF and mono-biotinylated BDNF, and both showed a stronger signal than the negative control condition. Then the biological activity of mono-biotinylated BDNF coupled to streptavidin quantum dots was evaluated to demonstrate that they can be used in live imaging experiments.
Cortical neurons were seeded in 10 millimeter covers and treated with a final concentration of 200 picomolar or two nanomolar BDNF quantum dots for 30 minutes before fixing and staining for phospho-CREB. CREB is a transcription factor which is targeted by activated ERK12 in cortical neurons. Stimulating neurons with increasing concentrations of BDNF quantum dots resulted in a dose-dependent increase of phosphorylation of CREB and presence of quantum dot particles surrounding the nucleus indicating that the BDNF quantum dot particles were endocytosed and triggered the activation of signaling pathways associated with BDNF-mediated TrkB activation.
A two-fold increase in phospho-CREB signal was detected when stimulating neurons with 200 picomolar BDNF quantum dots whereas stimulating with two nanomolar BDNF quantum dots resulted in a 3.5-fold increase in the phospho-CREB signal. Therefore, the biotinylated BDNF oviduct is biologically active and it doesn't lose its activity when coupled to streptavidin quantum dots making it suitable for immunofluorescence and live cell imaging. Finally, the imaging potential of BDNF quantum dots was evaluated in compartmentalized cultures using microfluidic chambers.
Cortical neurons were seeded in microfluidic chambers to separate the axonal and somatodendritic compartments and were stimulated with two nanomolar BDNF quantum dots for 3.5 hours. Live cell microscopy was performed and the resulting kymographs were used to quantify the speed of BDNF quantum dot containing organelles. An average moving speed of 0.91 micrometers per second was detected which is in line with previous analysis of cytoplasmic thymine-mediated transport.
Microfluidic chambers treated with two nanomolar streptavidin quantum dots didn't show moving quantum dots in the micro groups as shown by the kymograph. Cells grown under the same conditions were stimulated with 500 picomolar or two nanomolar BDNF quantum dots for 210 minutes and then fixed and labeled with a nuclear stain. Neurons treated with BDNF quantum dots show a dose-dependent accumulation of the labeled protein in all the analyzed subcompartments including the proximal and distal portions of the micro group and the somatodendritic compartment.
In contrast, control neurons showed almost no quantum dot signal throughout the chamber. Therefore, the BDNF quantum dot can be detected in live and fixed cells in microfluidic chambers. After watching this video, you should have a good understanding of how to produce mono-biotinylated BDNF oviduct in a chromatography column-based cost effective procedure.
