The authors gratefully acknowledge financial support from Fondecyt (1171137) (FCB), the Basal Center of Excellence in Science and Technology (AFB 170005) (FCB), Millenium-Nucleus (P07/011-F) (FCB), the Wellcome Trust Senior Investigator Award (107116/Z/15/Z) (GS) and a UK Dementia Research Institute Foundation award (GS). This work was supported by the Unidad de Microscop&#237;a Avanzada UC (UMA UC).

All experiments were carried out in accordance with the approved guidelines of CONICYT (Chilean National Commission for Scientific and Technological Research). The protocols used in this study were approved by the Biosecurity and Bioethical and Animal Welfare Committees of the Pontificia Universidad Cat&#243;lica de Chile. Experiments involving vertebrates were approved by the Bioethical and Animal Welfare Committee of the Pontificia Universidad Cat&#243;lica de Chile.
NOTE: The following prot...

<ol>
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In this article, an optimized methodology for the production and purification of mbtBDNF in an affinity chromatography-based procedure is described, based on the work of Sung and collaborators17. The optimizations include the use of a cost-effective transfection reagent (PEI) while maintaining the efficiency of more expensive transfection methods such as lipofectamine. This optimization translates into a significant cost reduction in the protocol, allowing for scalability while maintaining high co...

Neurons are the functional units of the nervous system possessing a complex and specialized morphology that allows synaptic communication, and thus, the generation of coordinated and complex behavior in response to diverse stimuli. Neuronal projections such as dendrites and axons are critical structural features involved in neuronal communication, and neurotrophins are crucial players in determining their morphology and function1. Neurotrophins are a family of secreted growth factors that include NGF, NT-3, NT-4, and brain-derived neurotrophic factor (BDNF)2. In the central nervous system (CNS), BDNF participates in diverse biological processes including neurotransmission, dendritic arborization, maturation of dendritic spines, long-term potentiation, among others3,4. Therefore, BDNF plays a critical role in regulating neuronal function.
Diverse cellular processes regulate BDNF dynamics and function. On the neuronal surface, BDNF binds the tropomyosin receptor kinase B (TrkB) and/or the p75 neurotrophin receptor (p75). BDNF-TrkB and BDNF-p75 complexes are endocytosed and sorted in different endocytic organelles5,6,7,8. Correct intracellular trafficking of the BDNF/TrkB complex is required for proper BDNF signaling in different neuronal circuits9,10,11. For this reason, a deep understanding of BDNF trafficking dynamics and its alterations found in pathophysiological processes is essential to understand BDNF signaling in health and disease. The development of novel and specific molecular tools to monitor this process will help to drive this field forward and allow a better grasp of the regulatory mechanisms involved.
There are several tools available for the study of BDNF trafficking in neurons. A commonly used methodology involves the transfection of recombinant BDNF tagged with fluorescent molecules such as green fluorescent protein (GFP) or the monomeric fluorescent red-shifted variant of GFP mCherry12,13. However, a major shortcoming of BDNF overexpression is that it eliminates the possibility of delivering known concentrations of this neurotrophin. Also, it may result in cellular toxicity, obscuring the interpretation of results14. An alternative strategy is the transfection of an epitope-tagged TrkB, such as Flag-TrkB. This methodology allows the study of TrkB internalization dynamics15, but it also involves transfection, which might result in altered TrkB function and cellular toxicity. To overcome these methodological hurdles, recombinant variants of NGF and BDNF containing an Avi sequence (BDNFAvi), which can be mono-biotinylated by the biotin-ligase enzyme BirA, were developed16,17. Biotinylated recombinant BDNF can be coupled to different streptavidin-bound tools, which include fluorophores, beads, paramagnetic nanoparticles among others for detection. In terms of live-cell imaging, quantum dots (QD) have become frequently used fluorophores, as they have desirable characteristics for single-particle tracking, such as increased brightness and resistance to photobleaching when compared to small molecule fluorophores18.
The production of mono-biotinylated BDNF (mbtBDNF) using BDNFAvi has been achieved by co-transfection of plasmids driving the expression of BDNFAvi and BirA, followed by the purification of the recombinant protein by affinity chromatography with a yield of 1-2 &#956;g of BDNF per 20 mL of HEK293-conditioned culture media17. Here, we propose a modification of this protocol that allows for BDNFAvi purification from 500 mL of HEK293-conditioned media, which seeks to maximize protein recovery in a chromatography-column based protocol for ease of manipulation. The used transfection agent, polyethyleneimine (PEI), ensures a cost-effective method without sacrificing transfection yield. The mono-biotinylation step has been adapted to an in vitro reaction to avoid the complications associated with co-transfections and to ensure homogeneous labeling of BDNF. The biological activity of the mbtBDNF was demonstrated by western blot and fluorescence microscopy experiments, including activation of pCREB and live cell imaging to study retrograde axonal transport of BDNF in microfluidic chambers. The use of this protocol allows for optimized, high-yield production of homogenous mono-biotinylated and biologically active BDNF.

<table border="0" cellpadding="0" cellspacing="0" style="width:665px;" width="665">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2 way stopcock</td>
			<td style="width:121px;">BioRad</td>
			<td style="width:161px;">7328102</td>
			<td style="width:167px;">Chromatography apparatus component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-mercaptoethanol</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">M6250</td>
			<td style="width:167px;">BDNF elution buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Acrylamide/Bisacrylamide</td>
			<td style="width:121px;">BioRad</td>
			<td style="width:161px;">1610154</td>
			<td style="width:167px;">SDS-PAGE gel preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Amicon Ultra-15 10K</td>
			<td style="width:121px;">Millipore</td>
			<td style="width:161px;">UFC901024</td>
			<td style="width:167px;">BDNF concentration</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ammonium Persulfate</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">A9164</td>
			<td style="width:167px;">SDS-PAGE gel preparation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">anti B-III-Tubulin antibody</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">T8578</td>
			<td style="width:167px;">Western blot assays for BDNF biological activity detection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti BDNF antibody</td>
			<td style="width:121px;">Alomone</td>
			<td style="width:161px;">AGP-021</td>
			<td style="width:167px;">Western blot assays for BDNF quantification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti BDNF antibody</td>
			<td style="width:121px;">Alomone</td>
			<td style="width:161px;">ANT-010</td>
			<td style="width:167px;">Western blot assays for BDNF quantification</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Anti ERK antibody</td>
			<td style="width:121px;">Cell Signaling</td>
			<td style="width:161px;">9102</td>
			<td style="width:167px;">Western blot assays for BDNF biological activity detection</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">anti pCREB antibody (S133)</td>
			<td style="width:121px;">Cell Signaling</td>
			<td style="width:161px;">9198</td>
			<td style="width:167px;">Western blot assays for BDNF biological activity detection</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">anti pERK antibody (T202, Y204)</td>
			<td style="width:121px;">Cell Signaling</td>
			<td style="width:161px;">4370</td>
			<td style="width:167px;">Western blot assays for BDNF biological activity detection</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">anti pTrkB antibody (Y515)</td>
			<td style="width:121px;">Abcam</td>
			<td style="width:161px;">ab109684</td>
			<td style="width:167px;">Western blot assays for BDNF biological activity detection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antibiotic/Antimycotic</td>
			<td style="width:121px;">Gibco</td>
			<td>15240-062</td>
			<td style="width:167px;">HEK293 maintenance</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ATP</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">A26209</td>
			<td style="width:167px;">BDNF monobiotinylation buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">B-27 Supplement</td>
			<td style="width:121px;">Gibco</td>
			<td>17504-044</td>
			<td style="width:167px;">Neuron maintenance</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bicine</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">B3876</td>
			<td style="width:167px;">BDNF monobiotinylation buffer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">BirA-GST</td>
			<td style="width:121px;">BPS Bioscience</td>
			<td style="width:161px;">70031</td>
			<td style="width:167px;">Enzyme for BDNF AviTag monobiotinylation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bovine Fetal Serum</td>
			<td style="width:121px;">HyClone</td>
			<td>HC.SH30396.02</td>
			<td style="width:167px;">HEK293 maintenance</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Bovine Serum Albumin</td>
			<td style="width:121px;">Jackson ImmunoResearch</td>
			<td>001-000-162</td>
			<td style="width:167px;">BDNF buffer modification component, blocking buffer for western blot and immunofluorescence</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">D-Biotin</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">B4639</td>
			<td style="width:167px;">BDNF monobiotinylation buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dithiothreitol</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:161px;">15508-013</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM High Glucose Medium</td>
			<td style="width:121px;">Gibco</td>
			<td>11965-092</td>
			<td style="width:167px;">Neuron seeding</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM Medium</td>
			<td style="width:121px;">Gibco</td>
			<td>11995-081</td>
			<td style="width:167px;">HEK293 maintenance</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Econo Column Funnel</td>
			<td style="width:121px;">BioRad</td>
			<td>7310003</td>
			<td style="width:167px;">Chromatography apparatus component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA</td>
			<td style="width:121px;">Merck</td>
			<td style="width:161px;">108418</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EZ-ECL Kit</td>
			<td style="width:121px;">Biological Industries</td>
			<td style="width:161px;">1633664</td>
			<td style="width:167px;">Protein detection by western blotting</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glutamax</td>
			<td style="width:121px;">Gibco</td>
			<td>35050-061</td>
			<td style="width:167px;">Neuron and HEK293 maintenance</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Glycerol</td>
			<td style="width:121px;">Merck</td>
			<td style="width:161px;">104094</td>
			<td style="width:167px;">BDNF elution buffer, lysis buffer for western blot assays</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Hettich Rotina 46R Centrifuge</td>
			<td style="width:121px;">Hettich</td>
			<td style="width:161px;">Discontinued</td>
			<td style="width:167px;">Centrifuge used for clearing the medium of debris</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Hettich Universal 32R Centrifuge</td>
			<td style="width:121px;">Hettich</td>
			<td style="width:161px;">Discontinued</td>
			<td style="width:167px;">Centrifuge used for protein concentrator centrifugation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Horse Serum</td>
			<td style="width:121px;">Gibco</td>
			<td>16050-122</td>
			<td style="width:167px;">Neuron seeding</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ImageQuant LAS 500</td>
			<td style="width:121px;">GE Healthcare Life Sciences</td>
			<td style="width:161px;">29005063</td>
			<td style="width:167px;">Western blot image acquisition</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Imidazole</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">I55513</td>
			<td style="width:167px;">BDNF buffer modification component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:121px;">Winkler</td>
			<td style="width:161px;">BM-1370</td>
			<td style="width:167px;">PBS component</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">KH2PO4</td>
			<td style="width:121px;">Merck</td>
			<td style="width:161px;">104873</td>
			<td style="width:167px;">PBS component</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Laminin</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:161px;">23017-015</td>
			<td style="width:167px;">Cover coating for compartmentalized neurons</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Luer Tubing Adaptor</td>
			<td style="width:121px;">BioRad</td>
			<td>7323245</td>
			<td style="width:167px;">Chromatography apparatus component</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Luminata&#8482; Forte Western HRP Substrate</td>
			<td style="width:121px;">Millipore</td>
			<td style="width:161px;">WBLUF0100</td>
			<td style="width:167px;">Protein detection by western blotting</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;">Mg(CH3COO)2</td>
			<td style="width:121px;">Merck</td>
			<td style="width:161px;">105819</td>
			<td style="width:167px;">BDNF monobiotinylation buffer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Mowiol 4-88</td>
			<td style="width:121px;">Calbiochem</td>
			<td style="width:161px;">475904</td>
			<td style="width:167px;">Mounting reagent for immunofluorescence assays</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MyOne C1 Streptavidin Magnetic Beads</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:161px;">65001</td>
			<td style="width:167px;">Biotinylation verification</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;">Na2HPO4</td>
			<td style="width:121px;">Merck</td>
			<td style="width:161px;">106586</td>
			<td style="width:167px;">BDNF buffer modification component</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">NaCl</td>
			<td style="width:121px;">Winkler</td>
			<td style="width:161px;">BM-1630</td>
			<td style="width:167px;">PBS component, BDNF buffer modification component</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;">NaH2PO4</td>
			<td style="width:121px;">Merck</td>
			<td style="width:161px;">106346</td>
			<td style="width:167px;">BDNF buffer modification component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Neurobasal Medium</td>
			<td style="width:121px;">Gibco</td>
			<td>21103-049</td>
			<td style="width:167px;">Neuron maintenance</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ni-NTA Agarose Beads</td>
			<td style="width:121px;">Qiagen</td>
			<td style="width:161px;">30210</td>
			<td style="width:167px;">BDNF AviTag purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nikon Ti2-E</td>
			<td style="width:121px;">Nikon</td>
			<td style="width:161px;"></td>
			<td style="width:167px;">Microscope for fluorescence imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nitrocellulose Membrane</td>
			<td style="width:121px;">BioRad</td>
			<td style="width:161px;">1620115</td>
			<td style="width:167px;">Protein transfer for western blotting</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">ORCA-Flash4.0 V3 Digital CMOS camera</td>
			<td style="width:121px;">Hamamatsu</td>
			<td style="width:161px;">C13440-20CU</td>
			<td style="width:167px;">Camera for epifluorescence imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">P8340 Protease Inhibitor Cocktail</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">P8340</td>
			<td style="width:167px;">BDNF buffer modification component</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Paraformaldehyde</td>
			<td style="width:121px;">Merck</td>
			<td style="width:161px;">104005</td>
			<td style="width:167px;">Fixative for immunofluorescence assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:121px;">Gibco</td>
			<td>15140-122</td>
			<td style="width:167px;">Neuron maintenance</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Poli-D-Lysine</td>
			<td style="width:121px;">Corning</td>
			<td style="width:161px;">DLW354210</td>
			<td style="width:167px;">Cover coating for compartmentalized neurons</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Poli-L-Lysine</td>
			<td style="width:121px;">Millipore</td>
			<td style="width:161px;">P2363</td>
			<td style="width:167px;">Cover coating for non-compartmentalized neurons</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Poly-Prep Chromatography Column</td>
			<td style="width:121px;">BioRad</td>
			<td>7311550</td>
			<td style="width:167px;">Chromatography apparatus component</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polyethyleneimine 25K</td>
			<td style="width:121px;">Polysciences Inc.</td>
			<td>PLY-0296</td>
			<td style="width:167px;">HEK293 transfection</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Quantum Dots 655 streptavidin conjugate</td>
			<td style="width:121px;">Invitrogen</td>
			<td style="width:161px;">Q10121MP</td>
			<td style="width:167px;">Monobiotinylated BDNF AviTag label for live and fixed cell experiments</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Saponin</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">S4521</td>
			<td style="width:167px;">Detergent for immunofluorescence assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sucrose</td>
			<td style="width:121px;">Merck</td>
			<td style="width:161px;">107687</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syldgard 184 silicone elastomer base</td>
			<td style="width:121px;">Poirot</td>
			<td style="width:161px;">4019862</td>
			<td style="width:167px;">Microfluidic chamber preparation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TEMED</td>
			<td style="width:121px;">Sigma</td>
			<td style="width:161px;">T9281</td>
			<td style="width:167px;">SDS-PAGE gel preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:121px;">Winkler</td>
			<td style="width:161px;">BM-2000</td>
			<td style="width:167px;">Lysis buffer component</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Triton X100</td>
			<td style="width:121px;">Merck</td>
			<td style="width:161px;">108603</td>
			<td style="width:167px;">Cell permeabilization in immunofluorescence and western blot assays</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin-EDTA 0.5%</td>
			<td style="width:121px;">Gibco</td>
			<td>15400-054</td>
			<td style="width:167px;">HEK293 passaging</td>
		</tr>
	</tbody>
</table>

an improved protocol to purify and directly mono-biotinylate recombinant bdnf in a tube for cellular trafficking studies in neurons

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A reproducible protocol was developed to directly&#160;mono-biotinylate BDNFAvi with the enzyme BirA in a tube. In this reaction, mono-biotinylated BDNFAvi retains its biological activity.
Neurotrophins are target-derived growth factors playing a role in neuronal development and maintenance. They require rapid transport mechanisms along the endocytic pathway to allow long-distance signaling between different neuronal compartments. The development of molecular tools to study the trafficking of neurotrophins has enabled the precise tracking of these proteins in the cell using in vivo recording. In this protocol, we developed an optimized and cost-effective procedure for the production of mono-biotinylated BDNF. A recombinant BDNF variant containing a biotinylable avi sequence (BDNFAvi) is produced in HEK293 cells in the microgram range and then purified in an easily scalable procedure using affinity chromatography. The purified BDNF can then be homogeneously mono-biotinylated by a direct in vitro reaction with the enzyme BirA in a tube. The biological activity of the mono-biotinylated BDNF (mbtBDNF) can be conjugated to streptavidin-conjugated to different fluorophores. BDNFAvi and mbtBDNF retain their biological activity demonstrated through the detection of downstream phosphorylated targets using western blot and activation of the transcription factor CREB, respectively. Using streptavidin-quantum dots, we were able to visualize mbtBDNF internalization concomitant with activation of CREB, which was detected with a phospho-CREB specific antibody. In addition, mbtBDNF conjugated to streptavidin-quantum dots was suitable for retrograde transport analysis in cortical neurons grown in microfluidic chambers. Thus, in tube produced mbtBDNF is a reliable tool to study physiological signaling endosome dynamics and trafficking in neurons.

The use of a chromatographic column-based protocol allows the processing of significant volumes of HEK293 conditioned media. In Figure 1, the results of the purification of BDNFAvi from 500 mL of conditioned media are shown. Consecutive elutions of BDNFAvi from the Ni-NTA agarose beads yield decreasing concentrations of BDNFAvi (Figure 1A). After four consecutive elutions (each lasting 15 min), the majority of the BDNF captured by the beads is recovered. The con...

Watch this Scientific Journal Video about An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons at JoVE.com

An Improved Protocol to Purify and Directly Mono-Biotinylate Recombinant BDNF in a Tube for Cellular Trafficking Studies in Neurons

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells in a cost-effective manner and is purified by affinity chromatography. BDNFavi is then directly mono-biotinylated with the enzyme BirA in a tube. BDNFavi and mono-biotinylated BDNFavi retain their biological activity when compared to commercially available BDNF.

Recombinant BDNF containing an Avi sequence (BDNFAvi) is produced in HEK293 cells and then cost-effectively purified by affinity chromatography. A ...

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.

an-improved-protocol-to-purify-directly-mono-biotinylate-recombinant

Research

JoVE Journal

Neuroscience

5.6K visualizações.  Pontificia Universidad Católica de Chile. O objetivo geral deste procedimento é produzir e purificar oviduct BDNF em um protocolo otimizado envolvendo transfecção de células HEK293 e purificação em uma coluna de cromatografia. O oviduto BDNF é então mono-biotiningado por uma reação in vitro para rotulagem e detecção posterior por microscopia de fluorescência. A principal vantagem deste procedimento é que ele permite a produção, purificação e mono-biotiningação de BDNF de forma otimizada.Os parâmetros de ge...