Nerve growth factor (ngf)

Manufactured by Santa Cruz Biotechnology

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NGF (Nerve Growth Factor) is a protein that plays a crucial role in the growth, maintenance, and survival of certain neurons in the body. It is a member of the neurotrophin family of growth factors. NGF is involved in the development and function of the nervous system.

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Lab products found in correlation

Brain derived neurotrophic factor (bdnf), by Santa Cruz Biotechnology (7 mentions) β actin, by Abcam (4 mentions) Gapdh, by Santa Cruz Biotechnology (2 mentions) Gapdh, by ABclonal (2 mentions) Ecl reagent, by Cytiva (1 mentions) Iblot dry blotting system, by Thermo Fisher Scientific (1 mentions) P nrf2, by Bioss Antibodies (1 mentions) P ampk, by Cell Signaling Technology (1 mentions) Protease inhibitor cocktail p8340, by Merck Group (1 mentions) Phosphatase inhibitor cocktail 1 2, by Merck Group (1 mentions) Ampkα, by Cell Signaling Technology (1 mentions) Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions) Xcell surelock mini cell, by Thermo Fisher Scientific (1 mentions) Novex tris glycine polyacrylamide gels, by Thermo Fisher Scientific (1 mentions) Sdf 1, by Abcam (1 mentions) P nf κb, by Cell Signaling Technology (1 mentions) Total nf κb, by Cell Signaling Technology (1 mentions) Total pi3k, by Cell Signaling Technology (1 mentions) Total erk, by Cell Signaling Technology (1 mentions) Phosphorylated akt p akt, by Cell Signaling Technology (1 mentions)

13 protocols using nerve growth factor (ngf)

Western Blot Analysis of Brain Proteins

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Sagittally cut hemi-brains brains were homogenized in ice-cold RIPA buffer (PBS, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS) with freshly added protease inhibitors (Protease Inhibitor Cocktail P8340 and, Phosphatase Inhibitor Cocktail 1&2, Sigma-Aldrich, St. Louis, MO; Phenylmethylsulfonylfluoride (PMSF), Fluka-Biochemica, Switzerland), incubated on ice for 30 min and centrifuged at 14,000 g for 10 min at 4°C. After centrifugation, the supernatant was collected and total protein concentrations were measured using the Bradford method. Tissue lysate was added to electrophoresis sample buffer and separated by SDS/PAGE under reducing conditions on a 12% separation gel. Proteins were transferred to nitrocellulose membranes using the iBlot dry-blotting system (Invitrogen Corp., Carlsbad, CA). Unspecific binding was blocked by incubation in 5% milk blocking buffer (PBS, 5% nonfat milk and 0.1% Tween 20). Membrane bound proteins were immunoblotted with antibodies to BDNF, NGF, NT3, Nrf2 (Santa Cruz Biotech, Santa Cruz, CA), p-AMPK, AMPKα (Cell Signaling, Beverly, MA), p-Nrf2 (BIOSS Antibodies, Woburn, MA) and β-actin (Abcam Inc., Cambridge, MA). Signals were developed using ECL reagent (Amersham Pharmacia Biotech, Buckinghamshire, England). Densities of the bands were evaluated using Image J software.

Allard J.S., Perez E.J., Fukui K., Carpenter P., Ingram D.K, & de Cabo R. (2015). Prolonged metformin treatment leads to reduced transcription of Nrf2 and neurotrophic factors without cognitive impairment in older C57BL/6J mice. Behavioural brain research, 301, 1-9.

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Western Blot Analysis of PC12 Cell Proteins

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Cells were cultured in the presence or absence of REAC for 24, 48, or 72 hours and for 7 days. Total cell lysates from PC12 were electrophoresed on 10% Novex Tris-glycine polyacrylamide gels (Invitrogen) in MOPS sodium dodecyl sulfate running buffer using an XCell SureLock™ Mini-Cell (Invitrogen) according to the instructions provided by the manufacturer. After protein transfer to polyvinylidenedifluoride membranes (Invitrogen), and membrane saturation and washing, immunoreaction was carried out for one hour at room temperature in the presence of the primary antibody, antisera against β Tubulin isotype III (Cell Signaling technology), NGF (Santa Cruz Biotechnology, Inc.), TH (Santa Cruz Biotechnology, Inc), Neurogenin (Santa Cruz Biotechnology, Inc), and GAPDH (Santa Cruz Biotechnology, Inc.) diluted to 1:1000. After additional washing, membranes were incubated with antirabbit (NGF ,GAPDH) or antimouse (β Tubulin isotype III) or antigoat (Neurogenin). Targeted protein expression was assessed using a chemoluminescence detection system (ECL Western blotting detection reagents were from Amersham Biosciences Corporation, Piscataway, NJ, USA).

Maioli M., Rinaldi S., Migheli R., Pigliaru G., Rocchitta G., Santaniello S., Basoli V., Castagna A., Fontani V., Ventura C, & Serra P.A. (2015). Neurological morphofunctional differentiation induced by REAC technology in PC12. A neuro protective model for Parkinson’s disease. Scientific Reports, 5, 10439.

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Protein Expression Analysis in Penis Tissue

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Penis tissue and MCECs were lysed in RIPA buffer (Sigma-Aldrich) Supplemented with protease inhibitors (GenDEPOT, Katy, TX, USA) and phosphatase inhibitors (GenDEPOT). Equal amounts of protein (40 µg/lane) were resolved by SDS-PAGE on 8% to 15% gels, and then transferred to polyvinylidene difluoride membranes. After blocking with 5% nonfat dry milk for 1 hour at room temperature, membranes were probed with antibodies against Hsp70 (Abcam; 1:1,000), neurotrophin-3 (NT-3; Santa Cruz Biotechnology; 1:1,000), nerve growth factor (NGF; Santa Cruz Biotechnology; 1:1,000), BDNF (Santa Cruz Biotechnology; 1:1,000), pro and cleaved caspase-3 (Novus Biologicals, Centennial, CO, USA; 1:1,000), phosphorylated-eNOS (p-eNOS; Invitrogen; 1:500), total eNOS (BD Biosciences, Franklin Lakes, NJ, USA; 1:500), phosphorylated-Akt (p-Akt; Cell Signaling; 1:1,000), total Akt (Cell Signaling; 1:1,000) p-PI3K (Cell Signaling; 1:1,000), total PI3K (Cell Signaling; 1:1,000) phosphorylatedp-ERK (p-ERK; Cell Signaling; 1:1,000), total ERK (Cell Signaling; 1:1,000), p-NF-κB (Cell Signaling; 1:500), total NF-κB (Cell Signaling; 1:500), Cse (Thermo Fisher Scientific; 1:1,000), SDF1 (Abcam; 1:1,000), HO-1 (Santa Cruz Biotechnology; 1:500), and/or β-actin (Abcam; 1:5,000), as described in the text. The results were quantified densitometrically using Image J software.

Ghatak K., Yin G.N., Hong S.S., Kang J.H., Suh J.K, & Ryu J.K. (2022). Heat Shock Protein 70 in Penile Neurovascular Regeneration Requires Cystathionine Gamma-Lyase. The World Journal of Men's Health, 40(4), 580-599.

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Western Blot Analysis of Neurotrophic Factors

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Equal amounts of protein (20 µg per lane) were electrophoresed on sodium dodecylsulfate-polyacrylamide gels (12% to 15%), transferred to nitrocellulose membranes, and probed with antibodies to NGF (Santa Cruz Biotechnology; 1:1000), BDNF (Santa Cruz Biotechnology; 1:1000), NT-3 (Santa Cruz Biotechnology; 1:1000), GAPDH (ABclonal, Woburn, MA, USA; 1:5000), or β-actin (Abcam; 1:6000). The results were quantified by densitometry.

Ghatak K., Yin G.N., Choi M.J., Limanjaya A., Minh N.N., Ock J., Song K.M., Kang D.H., Kwon Y.G., Kim H.M., Ryu J.K, & Suh J.K. (2017). Dickkopf2 rescues erectile function by enhancing penile neurovascular regeneration in a mouse model of cavernous nerve injury. Scientific Reports, 7, 17819.

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Neutralization of Growth Factors

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For this, antibodies against human GDNF, BDNF, NT-3, NGF, VEGF and IGF-1 (SantaCruz) were used. Around 1 mg/ml antibody was added to the CM and incubated with shaking for 4 hrs at room temperature.^{27 (link)}

Kumar A., Mishra H.K., Dwivedi P, & Subramaniam J.R. (2015). Secreted trophic factors of Human umbilical cord stromal cells induce differentiation and neurite extension through PI3K and independent of cAMP pathway. Annals of Neurosciences, 22(2), 97-106.

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Nanoparticle Effects on Neurotrophin Signaling

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Commercial grade titanium dioxide nanoparticles (TiO₂-NP) and titanium(IV) oxide fine particles (TiO₂-FP) were kindly provided by the National Institute for Occupational Safety and Health (NIOSH). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay kits were purchased from Cayman Chemical (Ann Arbor, MI); FITC Annexin-V Apoptosis Detection kits from BD Biosciences (San Jose, CA); and RNeasy kits for total RNA isolation from Qiagen (Valencia, CA). Primers for human NGF, BDNF, TrkA, TrkB and p75^NTR were purchased from SABiosciences (Valencia, CA) and primers for the β₂ microglobulin (B2M) housekeeping gene from RealTimePrimers (Elkins Park, PA).
The following primary antibodies, secondary antibodies, and isotype controls were used for immunostaining: NGF, BDNF, p75^NTR, GAPDH and JNK (Santa Cruz Biotechnology, Santa Cruz, CA); phospho-SAPK/JNK (Cell Signaling, Danvers, MA); pro-NGF (Millipore, Billerica, MA); TrkA and TrkB (R&D Systems, Minneapolis, MN); AlexaFluor 488-conjugated secondary antibodies (Invitrogen, Carlsbad, CA); and horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology). Western blotting reagents were purchased from Amersham Life Science (Buckinghamshire, United Kingdom). Interleukin 1 alpha (IL-1α) and IL-1 receptor antagonist (IL1-ra) were purchased from Santa Cruz Biotechnology.

Chakraborty S., Castranova V., Perez M.K, & Piedimonte G. (2017). Nanoparticles-Induced Apoptosis of Human Airway Epithelium is mediated by ProNGF/p75NTR Signaling. Journal of toxicology and environmental health. Part A, 80(1), 53-68.

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Protein Expression Analysis in Angiogenic Signaling

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Equal amounts of protein (40 µg per lane) were electrophoresed on sodium dodecylsulfate-polyacrylamide gels (8% to 15%), transferred to polyvinylidene difluoride membrane, and probed with antibodies to HGF (Santa Cruz Biotechnology, Delaware CA, USA; 1:1000), Ang1 (Novus Biologicals), Ang2 (Novus Biologicals; 1:1000), phospho-eNOS (Signaling, Beverly, MA, USA; 1:250), eNOS (Cell Signaling; 1:500), phospho-Akt (Cell Signaling; 1:1000), Akt (Cell Signaling; 1:1000), phospho-ERK (Cell Signaling; 1:1000), ERK (Cell Signaling; 1:1000), NGF (Santa Cruz Biotechnology; 1:1000), NT-3 (Santa Cruz Biotechnology; 1:1000), GAPDH (ABclonal, Woburn, MA, USA; 1:5000), or β-actin (Abcam; 1:6000). The results were quantified by densitometry (N = 4 per group).

Kwon M.H., Song K.M., Limanjaya A., Choi M.J., Ghatak K., Nguyen N.M., Ock J., Yin G.N., Kang J.H., Lee M.R., Gho Y.S., Ryu J.K, & Suh J.K. (2019). Embryonic stem cell-derived extracellular vesicle-mimetic nanovesicles rescue erectile function by enhancing penile neurovascular regeneration in the streptozotocin-induced diabetic mouse. Scientific Reports, 9, 20072.

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Antibody Characterization for Muscle Integrity

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The utilized primary polyclonal antibodies were directed against BDNF (code: sc 546, dilution: 1:1000) and NGF (code: sc 548, dilution 1:2000), both from Santa Cruz Biotechnology, Santa Cruz, CA, USA. The monoclonal antibody against intermediate filament protein desmin (code: M0760, dilution: 1:1000) from DAKO (Glostrup, Denmark) was used as a marker for myofibrillar integrity. All the used antibodies have previously been well characterized for their specificity.25, 26, 27, 28, 29, 30, 31

Shah F., Forsgren S., Holmlund T., Levring Jäghagen E., Berggren D., Franklin K.A, & Stål P. (2018). Neurotrophic factor BDNF is upregulated in soft palate muscles of snorers and sleep apnea patients. Laryngoscope Investigative Otolaryngology, 4(1), 174-180.

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Histopathological and Immunohistochemical Analysis of Skin Specimens

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Skin specimens in paraformaldehyde-fixed (4%) were embedded in paraffin for haematoxylin and eosin staining (H&E) and IHC. Mast cells were stained by Toluidine blue following the way of Puebla-Osorio et al. [23 (link)]. We assessed the histopathological alterations with computer-assisted quantitative image analysis in lesions, including epidermal thickness, area of Munro's microabscesses (MM), and the number of mast cells. Tissue sections were prepared for IHC with SP (Cat, No. sc-21715, 1 : 50, Santa Cruze), CGRP (Cat, No. sc-57053, 1 : 50, Santa Cruze), and NGF (Cat, No. sc-32300, 1 : 50, Santa Cruze) antibodies. The integrated optical density (IOD) of SP, CGRP, and NGF in the image was measured by Image Pro plus 6 software.

Li F., Han D., Wang B., Zhang W., Zhao Y., Xu J., Meng L., Mou K., Lu S., Zhu W, & Zhou Y. (2022). Topical Treatment of Colquhounia Root Relieves Skin Inflammation and Itch in Imiquimod-Induced Psoriasiform Dermatitis in Mice. Mediators of Inflammation, 2022, 5782922.

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Evaluating Protein Expression in SCs Treated with PRP

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Western blotting was performed to assess the changes in protein expression in human SCs treated with PRP at 0%, 5%, and 10% concentrations. The primary antibodies used were as follows: myelin-associated glycoprotein (MAG, Santa Cruz Biotechnology, Santa Cruz, CA), myelin basic protein (MBP, Santa Cruz Biotechnology, Santa Cruz, CA), neurotrophin-3 (NT-3, Santa Cruz Biotechnology, Santa Cruz, CA), nerve growth factor (NGF, Santa Cruz Biotechnology, Santa Cruz, CA), and extracellular signal-regulated kinase (ERK; Santa Cruz Biotechnology, Santa Cruz, CA). GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as an internal control. All blots derive from the same experiment and were processed in parallel. Quantitative analysis was performed using ImageJ software (version 1.49; Wayne Rasband, National Institutes of Health). To rule out the expression of NGF in rat PRP, NGF concentrations in SC-media were determined by ELISA (Abnova, Taipei, Taiwan) according to the manufacturer’s instructions.

Kim J.W., Kim J.M., Choi M.E., Jeon E.J., Park J.M., Kim Y.M., Choi S.H., Eom T., Shim B.S, & Choi J.S. (2022). Platelet-rich plasma loaded nerve guidance conduit as implantable biocompatible materials for recurrent laryngeal nerve regeneration. NPJ Regenerative Medicine, 7, 49.

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