The protocol describes the preparation of cell membrane affinity chromatography (CMAC) columns with immobilized cell membrane fragments containing functional transmembrane tropomyosin kinase receptor B proteins. The use of CMAC columns in the identification of specialized plant metabolites interacting with these receptors and present in complex natural mixtures is also explained.
Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, penicillin, paclitaxel, rapamycin, or artemisinin, commonly used in medical practice, have been first identified and isolated from natural products. However, the identification and isolation of biologically active specialized metabolites from natural sources is a challenging and time-consuming process. Traditionally, individual metabolites are isolated and purified from complex mixtures, following the extraction of biomass. Subsequently, the isolated molecules are tested in functional assays to verify their biological activity. Here we present the use of cellular membrane affinity chromatography (CMAC) columns to identify biologically active compounds directly from complex mixtures. CMAC columns allow for the identification of compounds interacting with immobilized functional transmembrane proteins (TMPs) embedded in their native phospholipid bilayer environment. This is a targeted approach, which requires knowing the TMP whose activity one intends to modulate with the newly identified small molecule drug candidate. In this protocol, we present an approach to prepare CMAC columns with immobilized tropomyosin kinase receptor B (TrkB), which has emerged as a viable target for drug discovery for numerous nervous system disorders. In this article, we provide a detailed protocol to assemble the CMAC column with immobilized TrkB receptors using neuroblastoma cell lines overexpressing TrkB receptors. We further present the approach to investigate the functionality of the column and its use in the identification of specialized plant metabolites interacting with TrkB receptors.
Botanical mixtures are rich in pharmacologically active compounds1, serving as a good source for the identification of new drug hits and leads2,3,4,5. The discovery of new medicines from natural products has been a fruitful approach and many currently approved drugs originated from compounds first identified in nature. The chemical diversity of natural compounds is hard to be matched by man-made libraries of chemically synthesized molecules. Many natural compounds interact with and modulate human protein targets and can be considered evolutionarily optimized drug-like molecules6. These natural compounds are particularly well suited for drug lead identification to use in neurological disorders6. Two of the currently FDA-approved drugs for the management of Alzheimer's disease (AD) are derived from natural alkaloids, namely: galantamine and rivastigmine (a derivative of physostigmine)6. L-DOPA, presently the most commonly prescribed drug for Parkinson's disease, was first identified from the broad bean (Vicia faba L.)7. Pergolide and lisuride, dopaminergic receptor agonists are the derivatives of natural ergot alkaloids from the parasitic fungus Claviceps purpurea8. Reserpine, an alkaloid isolated from Indian snakeroot (Rauvolfia serpentina (L.) Benth. ex Kurz) was one of the first antipsychotic drugs9. Recently, dysregulated immune response and systemic inflammation have been linked to the development of numerous neurological ailments, such as major depressive disorder or neurodegenerative diseases10. A plant-based diet together with other lifestyle interventions has been found to improve cognitive and functional abilities in the elderly11,12,13,14,15,16,17,18,19,20,21. Certain electrophilic molecules belonging to triterpenes and polyphenols have been found to modulate inflammatory responses in both in vitro and in vivo models12. For instance, natural compounds containing α,β-unsaturated carbonyl (e.g., curcumin, cinnamaldehyde), or isothiocyanate group (e.g., sulforaphane) interfere with Toll-like receptor-4 (TLR4) dimerization inhibiting the downstream synthesis of pro-inflammatory cytokines in a murine interleukin-3 dependent pro-B cell line12,22. Epidemiological evidence points strongly that dietary phytochemicals, present in complex food matrices, may also constitute a viable source of new drug leads6.
One of the major obstacles in the identification of biologically active molecules present in plant extracts, including plant-based food, is the complexity of the investigated samples. Traditionally, the individual compounds are isolated, purified, and subsequently tested for biological activity. This approach usually leads to the identification of the most abundant and well-characterized compounds. Phenotypic drug discovery approaches without a defined molecular target rely on the bio-guided-fractionation of complex mixtures23. In this approach, an extract is fractionated into less complex sub-fractions that are subsequently tested in phenotypic assays. The isolation and purification of active compounds are guided by biological activity verified in the assay. The knowledge of the identity of a specified drug target may significantly speed up the identification of pharmacologically active compounds present in complex mixtures. Those approaches are usually based on the immobilization of the molecular target, for example, an enzyme, on a solid surface, like magnetic beads23. The immobilized targets are subsequently used in the screening experiments resulting in the isolation of compounds interacting with the target. While this approach has been extensively used in the identification of compounds targeting cytosolic proteins, it has been less commonly applied in the identification of chemicals interacting with transmembrane proteins (TMPs)23. An additional challenge in the immobilization of TMPs stems from the fact that the activity of the protein depends on its interaction with cell membrane phospholipids and other molecules in the bilayer such as cholesterol23,24. It is important to preserve these subtle interactions between proteins and their native phospholipid bilayer environment when attempting to immobilize the transmembrane target.
In cellular membrane affinity chromatography (CMAC) cell membrane fragments, and not purified proteins, are immobilized on the artificial membrane (IAM) stationary phase particles23. IAM stationary phases are prepared by covalently bonding phosphatidylcholine analogs onto silica. Recently novel classes of IAM stationary phases have been developed in which free amine and silanol groups are end-capped (IAM.PC.DD2 particles). During CMAC columns preparation cell membrane fragments are immobilized onto the surface of IAM particles through adsorption.
CMAC columns have been used to date to immobilize different classes of TMPs including ion channels (e.g., nicotinic receptors), GPCRs (e.g., opioid receptors), protein transporters (e.g., p-glycoprotein), etc.24. The immobilized protein targets have been used in the characterization of pharmacodynamics (e.g., dissociation constant, Kd) or determining binding kinetics (kon and koff) of small molecule ligands interacting with the target as well as in the process of identification of potential new drug leads present in complex matrices24. Here we present the preparation of CMAC columns with the immobilized tropomyosin kinase receptor B (TrkB), which has emerged as a viable target for drug discovery for numerous nervous system disorders.
Previous studies showed that the activation of the brain-derived neurotrophic factor (BDNF)/TrkB pathway is associated with the improvement of certain neurological ailments, such as AD or major depressive disorder25,26,27,28. It was reported that BDNF levels and its receptor TrkB expression decrease in AD, and similar reductions impair hippocampal function in animal models of AD29. Decreased levels of BDNF were reported in serum and brain of AD patients30,31,32. Tau overexpression or hyperphosphorylation were found to down-regulate BDNF expression in primary neurons and AD animal models33,34,35. Additionally, BDNF was reported to have protective effects on β-amyloid induced neurotoxicity in vitro and in vivo36. Direct BDNF administration into the rat brain was shown to increase learning and memory in cognitively impaired animals37. BDNF/TrkB emerged as a valid target for ameliorating neurological and psychiatric disorders including AD28,38. Targeting the BDNF/TrkB signaling pathway for the development of therapies in AD will potentially enhance our understanding of the disease39. Unfortunately, BDNF itself cannot be used as a treatment because of its poor pharmacokinetic properties and adverse side effects40. Small molecule activators of TrkB/BDNF pathways have been explored as potential TrkB ligands41,42,43. Among tested small molecule agonists, 7,8-dihydroxyflavone (7,8-DHF), has been shown to activate the BDNF/TrkB pathway41,44,45,46. A derivative of 7,8-DHF (R13; 4-Oxo-2-phenyl-4H-chromene-7,8-diyl bis(methylcarbamate)) is currently under consideration as a possible drug for AD47. Recently, it was shown that several antidepressants work through directly binding to TrkB and promoting BDNF signaling, further stressing the importance of pursuing TrkB as a valid target to treat various neurological disorders48.
The protocol describes the process of assembling functional TrkB column and TrkB-NULL negative control column. The columns are characterized using a known natural product small-molecular ligand: 7,8-DHF. Additionally, we describe the process of screening complex matrices, using plant extract as an example, for the identification of compounds interacting with TrkB.
1. Cell culture of SH-SY5Y neuroblastoma cells (TrkB and TrkB-NULL (parental) cell lines)
NOTE: Cell lines (SH-SY5Y Cell Line (TrkB, BR6) and SH-SY5Y Parental Cell Line (TrkB NULL))49,50 were purchased from Kerafast. Cultured cells are used as a source of the transmembrane receptors to be immobilized for the preparation of CMAC columns. The following steps describe how to obtain cell membrane fragments and assemble functional CMAC columns.
2. Cell harvesting
3. Cell homogenization
4. Cell membrane solubilization
5. Cell membrane immobilization on IAM.PC.DD2 particles
6. CMAC column packing
7. CMAC column characterization
8. Missing peak chromatography approach to identify potential TrkB binders from gotu kola extract
Following the protocol, two CMAC chromatographic columns were assembled: one with the immobilized SH-SY5Y neuroblastoma cell membrane fragments with overexpressed TrkB and one with SH-SY5Y TrkB-NULL cell membrane fragments. The correctly assembled CMAC column is presented in Figure 1 and the steps involved in cell membrane fragment immobilization are presented in Figure 2.
Since the immobilization of TrkB receptors on IAM.PC.DD2 chromatographic stationary phase had not been attempted before, the successful immobilization of the receptors was confirmed by antibody staining and frontal affinity chromatography using a marker ligand: 7,8-DHF. A schematic representation of the antibody staining experiment is presented in Figure 2. Cell membrane fragments obtained from neuroblastoma cell line overexpressing TrkB receptors and cell membrane fragments from parental cell line without TrkB receptors (TrkB-NULL)) were immobilized on IAM.PC.DD2 particles using the optimized protocol. Subsequently, the particles with immobilized cell membrane fragments were incubated with BDNF (physiological ligand) and then with primary and fluorescent-labeled secondary antibodies (Figure 2). IAM.PC.DD2 particles with immobilized neuroblastoma TrkB cell membrane fragments and in the presence of BDNF resulted in fluorescently labeled particles (Figure 3C). No fluorescence (except for weak background fluorescence) was observed when TrkB-NULL cell membrane encapsulated IAM particles were investigated (Figure 3A) or when no BDNF was used in the case of TrkB cell membrane encapsulated IAM particles (Figure 3B).
To characterize the binding of a small marker ligand (7,8-DHF) to the immobilized TrkB receptors frontal affinity chromatography was performed with different concentrations of 7,8-DHF. A typical chromatogram of increasing 7,8-DHF concentrations on a functional CMAC column is presented in Figure 4. Specific binding of 7,8-DHF to the immobilized TrkB was confirmed by the concentration-dependent decreases in the retention time of the marker ligand. Frontal affinity chromatography results obtained on a non-functional CMAC column are presented in Figure 5. The lack of concentration-dependent changes in retention of the marker ligand indicates the unsuccessful attempt in CMAC preparation.
Compounds injected onto the CMAC column interact not only specifically but can also non-specifically interact with IAM.PC.DD2 particles, phospholipid bilayer of the immobilized cell membrane fragments, and other proteins present in the bilayer. To rule out compounds non-specifically interacting with the CMAC column during the process of screening of complex extracts for TrkB binders it is important to prepare CMAC negative control column by immobilizing cell membrane fragments of the parental cell line not expressing the targeted protein (in this case TrkB-NULL). Frontal affinity chromatography results obtained on a negative CMAC TrkB-NULL column are presented in Figure 6.
Functional CMAC columns can be used for different purposes, such as characterization of the targeted protein, studying interactions of individual compounds with the immobilized targets (e.g., obtaining dissociation constant, Kd) or determining binding kinetics (kon and koff) etc.24. The columns can also be used to screen complex samples, such as plant extracts for the presence of compounds binding to TrkB receptors, therefore potentially containing molecules that act as TrkB agonists or antagonists. To verify if an extract potentially contains compounds interacting with the immobilized receptors a simple displacement experiment is performed. The result of the competition experiment performed with gotu kola extract and 500 nM of 7,8-DHF on a functional TrkB column is presented in Figure 7. The addition of 0.2% aqueous gotu kola extract (10 mg/mL) resulted in a significant reduction of 7,8-DHF retention indicating the presence of competing ligand(s) for the agonist binding site. The result of the displacement experiment performed on the CMAC negative TrkB-NULL column is presented in Figure 8. The lack of reduction of 7,8-DHF retention on that column further confirms the lack of functional TrkB receptors on the CMAC TrkB-NULL column.
To identify specialized metabolites specifically interacting with the immobilized targets, CMAC columns are used in an approach called missing-peak chromatography52 following the displacement experiment. In this approach, a small volume of the investigated extract is chromatographed in parallel on the column containing the investigated immobilized target and CMAC negative control column. These two columns differ in the expression of the target, therefore any differences in the retention patterns of individual molecules are due to the specific nature of interactions with the investigated targets on the CMAC column containing these TMPs. Timed fractions from both columns are collected, concentrated, and subsequently analyzed on a C18 column. The obtained chromatograms are compared, and compounds represented by peaks similarly retained on both columns are labeled as non-specifically interacting molecules. The presence of a peak in later fractions on the CMAC column with the immobilized receptors and lack of a peak representing the same compound in early fractions of CMAC negative column represents specific interaction of the compound with the immobilized target. The compounds identified in this step can be targeted for isolation and further testing, without the need to perform bio-guided fractionation. The missing-peak chromatography approach was used to identify compounds specifically interacting with TrkB receptors from gotu kola extract. Gotu kola extract was fractionated on both TrkB and TrkB-NULL columns and compounds present in these fractions were analyzed using UPLC-QTOF-MS. Investigation of the chromatograms of each of the fractions led to the identification of a compound strongly retained on TrkB column (50-55 min fraction), while eluting early on TrkB-NULL column (0-5 min fraction), indicating specific interaction with the immobilized TrkB receptors (Figure 9). The compound eluting at ~ 17.48 min (Figure 9) is now targeted for isolation and testing in functional assays.
Figure 1. Correctly assembled CMAC column. Please click here to view a larger version of this figure.
Figure 2. CMAC preparation steps and fluorescent antibody labeling of the immobilized receptors. Schematic representation of the preparation of a CMAC column (1-4) and the antibody labeling experiment (5-8). (1) Homogenized cell membrane fragments containing targeted transmembrane protein, TrkB. (2) Solubilized cell membrane fragments in a micellar structure. (3) Cell membrane fragments immobilized on the surface of the IAM particles. (4) IAM particles with immobilized cell membrane fragments packed into a glass column. (5) Immobilized TrkB receptor in the cell membrane fragments. (6) Binding of the natural ligand BDNF to the functional TrkB receptor. (7) Binding of the primary antibodies to BDNF molecules. (8) Binding of fluorophore-labeled secondary antibodies to the primary antibodies resulting in green fluorescence. Please click here to view a larger version of this figure.
Figure 3. Immobilization of cell membrane fragments with TrkB receptors onto IAM particles. Confocal microscopy images showing the immobilization of cell membrane fragments with functional TrkB receptors on IAM particles. (A) Cell membrane fragments from SH-SY5Y TrkB-NULL cell line immobilized on IAM particles after incubation with BDNF, primary antibody, and fluorophore-labeled secondary antibody. (B) Cell membrane fragments from SH-SY5Y Neuroblastoma cell lines expressing TrkB immobilized on IAM particles after incubation with primary antibody and fluorophore-labeled secondary antibody without BDNF. (C) Cell membrane fragments from SH-SY5Y neuroblastoma cell lines expressing TrkB immobilized on IAM particles after incubation with BDNF, primary antibody, and fluorophore-labeled secondary antibody. Please click here to view a larger version of this figure.
Figure 4. Frontal affinity chromatograms of 7,8-DHF on the TrkB CMAC column. Frontal chromatogram of increasing concentrations of 7,8-DHF on the TrkB CMAC column, where A – 1 mM, B – 750 nM, C – 500 nM, and D – 300 nM. Ammonium acetate buffer (10 mM, pH 7.4) with 5% methanol was used as eluent at a flow rate of 0.4 mL/min. Please click here to view a larger version of this figure.
Figure 5. Frontal affinity chromatograms of 7,8-DHF on nonfunctional TrkB CMAC column. Frontal chromatogram of different concentrations of 7,8-DHF on the non-functional TrkB CMAC column, where A -1 μM, B – 1 μM, C – 750 nM, and D – 500 nM. Ammonium acetate buffer (10 mM, pH 7.4) with 5% methanol was used as eluent at a flow rate of 0.4 mL/min. Please click here to view a larger version of this figure.
Figure 6. Frontal affinity chromatograms of 7,8-DHF on the TrkB-NULL CMAC column. Frontal chromatogram of increasing concentrations of 7,8-DHF on the TrkB-NULL CMAC column, where A – 1 mM, B – 750 nM, C – 500 nM, and D – 300 nM. Ammonium acetate buffer (10 mM, pH 7.4) with 5 % methanol was used as eluent at a flow rate of 0.4 mL/min. Please click here to view a larger version of this figure.
Figure 7. Frontal affinity chromatograms of 7,8-DHF with 0.2% gotu kola extract on the TrkB CMAC column. Representative frontal elution profile of (A) 500 nM 7,8-DHF + 0.2% gotu kola extract (10 mg/ml) and (B) 500 nM 7,8-DHF on the CMAC TrkB column. Ammonium acetate buffer (10 mM, pH 7.4) with 5% methanol was used as eluent at a flow rate of 0.4 mL/min. Please click here to view a larger version of this figure.
Figure 8. Frontal affinity chromatograms of 7,8-DHF with 0.2% gotu kola extract on the TrkB-NULL CMAC column. Representative frontal elution profile of (A) 500 nM 7,8-DHF and (B) 500 nM 7,8-DHF + 0.2% gotu kola extract (10 mg/mL) on the TrkB-NULL CMAC column. Ammonium acetate buffer (10 mM, pH 7.4) with 5% methanol was used as eluent at a flow rate of 0.4 mL/min. Please click here to view a larger version of this figure.
Figure 9. UPLC-MS chromatograms of gotu kola extract fractions. UPLC-MS chromatograms of gotu kola extract fractions (A) eluted between 0-5 min from TrkB-NULL CMAC column and (C) eluted between 50-55 min from CMAC TrkB column. A compound with a retention time of 17.48 min present in both fractions, as confirmed by matching mass spectra (B and D) was identified as a potential TrkB binder. Please click here to view a larger version of this figure.
Homogenization Buffer | ||||
1 | Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) | 50 mM | ||
2 | Sodium chloride (NaCl) | 100 mM | ||
3 | Calcium chloride anhydrous (CaCl2) | 3 mM | ||
4 | Magnesium chloride hexahydrate (MgCl2) | 2 mM | ||
5 | Potassium chloride (KCl) | 5 mM | ||
6 | Phenylmethanesulfonyl fluoride (PMSF) | 0.1 mM | ||
7 | Benzamidine | 3 mM | ||
8 | Protease inhibitor cocktail (100X) | (1/100) | ||
9 | Glycerol | 10% | ||
10 | Adenosine 5'-triphosphate (ATP) disodium salt hydrate | 100 µM | ||
11 | Ethylenediaminetetraacetic acid (EDTA) | 5mM |
Table 1. Homogenization buffer composition.
Solubilization Buffer | ||||
1 | Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) | 50 mM | ||
2 | Sodium chloride (NaCl) | 100 mM | ||
3 | Calcium chloride anhydrous (CaCl2) | 3 mM | ||
4 | Magnesium chloride hexahydrate (MgCl2) | 2 mM | ||
5 | Potassium chloride (KCl) | 5 mM | ||
6 | Phenylmethanesulfonyl fluoride (PMSF) | 0.1 mM | ||
7 | Benzamidine | 3 mM | ||
8 | Protease inhibitor cocktail (100X) | (1/100) | ||
9 | Glycerol | 10% | ||
10 | Adenosine 5'-triphosphate (ATP) disodium salt hydrate | 100 µM | ||
11 | Sodium cholate | 2% |
Table 2. Solubilization buffer composition.
Dialysis Buffer | ||||
1 | Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) | 50 mM | ||
2 | Sodium chloride (NaCl) | 100 mM | ||
3 | Calcium chloride anhydrous (CaCl2) | 0.1 mM | ||
4 | Ethylenediaminetetraacetic acid (EDTA) | 5 mM | ||
5 | Phenylmethanesulfonyl fluoride (PMSF) | 0.1 mM |
Table 3. Dialysis buffer composition.
Identification of active compounds present in complex mixtures of specialized metabolites is a very challenging task23. Traditionally, individual compounds are isolated, and their activity is tested in different assays. This approach is time-consuming and costly and often leads to isolation and identification of the most abundant and well-characterized compounds23. Currently used high-throughput screening assays rely heavily on screening combinatorial chemistry libraries with already known targets and are not designed to identify and isolate biologically active compounds present in complex mixtures53.
CMAC allows for the identification of compounds targeting TMPs23,24,54. In this technique cell membrane fragments with TMPs are immobilized on the surface of IAM stationary phase particles23,24. CMAC is a unique approach that allows for the immobilization of cell membrane fragments with targeted proteins on the solid support imitating cell membrane phospholipid bilayer24. This allows preserving the functionality of immobilized protein targets and provides close to physiological conditions while using CMAC to identify small molecules interacting with TMPs. CMAC offers the advantage of opportunities for the discovery of novel chemical scaffolds from unique natural products libraries, that cannot be tested using any other of the currently used assays23. The proposed approach allows for the identification of compounds interacting with TMPs without unraveling the nature of this interaction. The mode of interaction (allosteric or orthosteric interaction; inhibition or activation) needs to be verified using functional cell-based assays. CMAC with the immobilized protein targets can also be used in the characterization of pharmacodynamics (e.g. dissociation constant, Kd) or determining binding kinetics (kon and koff) of small molecule ligands interacting with the target as well as in the process of characterization of the binding sites on the immobilized protein24. Although CMAC only allows identification of compounds binding to TMPs, it is an innovative approach that significantly speeds up the first step in the drug discovery pipeline, as it does not require compound purification, which has been currently the major bottleneck in the drug discovery process from natural mixtures.
Although the presented protocol focuses on the immobilization of one transmembrane receptor (TrkB), CMAC technology can be utilized in the preparation of columns with other targets. One of the crucial aspects of CMAC preparation is the choice of cell line or tissue that is used to isolate cell membrane fragments. If columns are used in the screening of complex mixtures for compounds interacting with the immobilized receptors, it is advised to use cell lines overexpressing the targeted protein. Using such a cell line will result in a higher number of immobilized targets and increase the chance of identification of compounds with a lower affinity towards the target or less abundant molecules. If one focuses on the characterization of the immobilized protein, the use of a native cell line is recommended, as post-translational modifications the protein undergoes as well as the phospholipid environment may significantly differ in the transfected cell line.
Some aspects of cell membrane isolation and immobilization on IAM particles are critical and may require modifications depending on the receptor type or the source of the protein. To prevent proteolytic cleavage of the immobilized proteins, it is essential to use proper protease inhibitors in the homogenization and solubilization buffers. A literature search is recommended to identify required protease inhibitors. In the process of protocol optimization, we determined that the addition of ATP and glycerol increases the number of binding sites (Bmax) on CMAC columns when immobilizing TrkB. Other cofactors may be necessary when optimizing the immobilization of other types of transmembrane targets24. Currently, we are investigating the addition of cholesterol in the process of TrkB immobilization, as recently cholesterol was found to modify the effects of TrkB ligands48. It was previously reported that the addition of cholesterol and/or other lipids may be required for obtaining functional CMAC columns24.
Different types of detectors may be used to monitor the column eluate including diode array detector for ligands with chromophores, mass spectrometry, or radio flow detector for radioactive ligands.
Monitoring the stability of CMAC columns is of critical importance. CMAC columns may be stable for up to several months, depending on the type of immobilized protein, frequency of use, and storage conditions. It is recommended to store the column in 0.05% sodium azide solution in ammonium acetate buffer at 4 °C if the column remains unused for more than a week. It is important to thoroughly wash the column with ammonium acetate buffer after longer periods before attempting to use it. It is advised to monitor the functionality of the column by injecting a selected concentration of a marker ligand weekly.
Despite numerous advantages, CMAC technology has several limitations that need to be taken into consideration when using the columns in drug discovery endeavors. Firstly, the number of immobilized transmembrane targets decreases with time, and therefore it is recommended that the total number of binding sites is monitored weekly24. One of the reasons for this decrease is the consequence of the immobilization process that is based on adsorption and does not involve the introduction of covalent bonds. Lipophilic compounds may be strongly retained on CMAC columns due to significant nonspecific binding to the IAM surface and phospholipid bilayers. This significantly increases the analysis time decreasing throughput. The process of CMAC column preparation is cell line-specific and requires a thorough understanding of the nature of immobilized proteins, making it less suitable for less characterized targets.
The authors have nothing to disclose.
Z.C.A. was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) 2219- International Postdoctoral Research Fellowship Program. Research reported in this publication was supported by the National Center for Complimentary and Integrative Medicine of the National Institutes of Health under award number 1R41AT011716-01. This work was also partially supported by American Society of Pharmacognosy Research Starter Grant, Regis Technologies grant to L.C. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
7-8 Dihydroxyflavone hydrate | Sigma-Aldrich | D5446-10 mg | ≥98% (HPLC) |
Adenosine 5'-triphosphate (ATP) disodium salt hydrate | Sigma-Aldrich | A2383-1 g | |
Ammonium acetate | VWR Chemicals BDH | BDH9204-500 g | |
BDNF antibody | Invitrogen | PA5-15198-400 μL | Primary antibody; 2 mg/mL of concentration |
Benzamidine hydrochloride hydrate | Sigma-Aldrich | B6506-25 g | |
Brain derived neurotrophic factor (BDNF) human | Sigma-Aldrich | B3795-10 μg | Recombinant, expressed in E. coli, lyophilized powder, suitable for cell culture |
Calcium chloride | VWR Analytical | BDH9224-1 kg | |
Cholic acid sodium salt | Alfa Aesar | J62050-100 g | |
Dounce homogenizer | VWR | 71000-516 | 40 mL, 285 mm (overall lenght), 32 x 140 mm (O.D. x H) |
Ethanol | Sigma-Aldrich | 493511 | |
Ethylenediaminetetraacetic acid (EDTA) | VWR Analytical | BDH-9232-500 g | |
Fetal bovine serum | Sigma-Aldrich | F2442-500 mL | sterile-filtered, suitable for cell culture |
G418 disulfate salt solution | Sigma-Aldrich | G8168-100 mL | 50 mg/mL in H2O, 0.1 μm filtered, suitable for cell culture |
Glycerol | VWR Life Science | E520-100 mL | |
Immobilized artificial membrane (IAM.PC.DD2) | Regis Technologies, Inc. | 1-771050-500 | |
Magnesium chloride hexahydrate | VWR Analytical | BDH9244-500 mL | |
Methanol | Sigma-Aldrich | 322425 | |
Nikon Plan Fluor | Nikon | Confocal laser scanning microscope | |
Normal goat serum (10%) | Life Technologies | 50197Z | |
Penicillin-Streptomycin | Sigma-Aldrich | P4333-100 mL | |
Phenylmethanesulfonyl fluoride (PMSF) | Thermo Scientific | 36978-5 g | |
Phosphate buffered saline (PBS) | VWR Life Science | K812-500 mL | 1x |
Potassium chloride | VWR Chemicals BDH | 0395-1 kg | |
Protease inhibitor cocktail | VWR Life Science Ambreso | M221-1 mL | Proteomics grade, containing 50 mM AEBSF, 30 µM aprotonin, 1 mM bestatin, 1 mM E-64 and 1 mM leupeptin |
RPMI-1640 medium | Sigma-Aldrich | R8758-500 mL | with L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture |
Secondary antibody goat anti-rabbit IgG (H+L) | Invitrogen Alexa Flour Plus 488 | A32731 | |
SH-SY5Y Neuroblastoma cell lines expressing Trk-B | Kerafast | ECP007 | |
SH-SY5Y Trk-NULL cell line | Kerafast | ECP005 | |
Snake skin dialysis tubing | Thermo Scientific | 88245 | 10K MWCO, 35 mm dry I.D. |
Sodium azide | Sigma-Aldrich | S2002 | |
Sodium chloride | BDH VWR Analytical | BDH9286-2.5 kg | |
Tricorn 5/20 column | GE Healthcare | 24-4064-08 | |
Tris-HCl | VWR Life Science | 0497-1 kg | |
Trypsin-EDTA solution | Sigma-Aldrich | T4049-500 mL | 0.25%, sterile-filtered, suitable for cell culture, 2.5 g porcine trypsin and 0.2 g EDTA |