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Recombinant Human MANF protein

Cat.No. : MANF-536H
Product Overview : Recombinant Human MANF protein was expressed in Escherichia coli.
Availability February 10, 2025
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Description : MANF is a secreted neurotrophic factor that is expressed in brain, neuronal and certain non-neuronal tissues. It has been shown to promote survival, growth and function of dopamine specific neurons. MANF and its structural homolog CDNF, each contain an N-terminal saposin-like lipid binding domain, and a carboxyl-terminal domain, which is not homologous to previously characterized protein structures. MANF and CDNF can prevent 6-OHDA induced degeneration of dopaminergic neurons by triggering survival pathways in a rat experimental model of Parkinson disease. Mature human MANF is 99 %, 98 % and 96 % a.a. identical to mature rat, mouse and bovine MANF respectively.
Source : E.coli
Species : Human
Form : Lyophilized from a 0.2μm filtered concentrated solution in PBS, pH 7.4.
Bio-activity : Fully biologically active when compared to standard. The ED50 as determined by a cell proliferation assay using rat C6 cells is less than 20 μg/ml, corresponding to a specific activity of > 50 IU/mg.
Molecular Mass : Approximately 18.2 kDa, a single non-glycosylated polypeptide chain containing158 amino acids.
Protein length : 158
AA Sequence : LRPGDCEVCISYLGRFYQDLKDRDV TFSPATIENELIKFCREARGKENRL CYYIGATDDAATKIINEVSKPLAHH IPVEKICEKLKKKDSQICELKYDKQ IDLSTVDLKKLRVKELKKILDDWGE TCKGCAEKSDYIRKINELMPKYAPK AASARTDL
Endotoxin : Less than 1 EU/µg of rHuMANF as determined by LAL method.
Purity : >95% by SDS-PAGE and HPLC analysis.
Storage : Use a manual defrost freezer and avoid repeated freeze-thaw cycles. 12 months from date of receipt, -20 to -70 centigrade as supplied. 1 month, 2 to 8 centigrade under sterile conditions after reconstitution. 3 months, -20 to -70 centigrade under sterile conditions after reconstitution.
Reconstitution : We recommend that this vial be briefly centrifuged prior to opening to bring the contents to the bottom. Reconstitute in sterile distilled water or aqueous buffer containing 0.1 % BSA to a concentration of 0.1-1.0 mg/mL. Stock solutions should be apportioned into working aliquots and stored at ≤-20 centigrade. Further dilutions should be made in appropriate buffered solutions.
Tag : Non
Gene Name : MANF
Official Symbol : MANF
Synonyms : MANF; mesencephalic astrocyte-derived neurotrophic factor; arginine rich, mutated in early stage tumors , ARMET; ARP; arginine-rich, mutated in early stage tumors; ARMET; MGC142148; MGC142150;
Gene ID : 7873
mRNA Refseq : NM_006010
Protein Refseq : NP_006001
MIM : 601916
UniProt ID : P55145

Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) Regulates Neurite Outgrowth Through the Activation of Akt/mTOR and Erk/mTOR Signaling Pathways

Journal: Frontiers in Molecular Neuroscience    PubMed ID: 33071755    Data: 2020/9/24

Authors: Wen Wen, Yongchao Wang, Jia Luo

Article Snippet:The following cells and materials were used: N2a (CCL-131), SH-SY5Y (CRL-2266) and HEK293 (CRL-1573) cells were from ATCC (Manassas, VA, United States); MEM (11095-080), high glucose DMEM (10569-010), L-methionine free DMEM (21013-024), FBS, antibiotic-antimycotic (15240112), GeneArt Genomic Cleavage Detection Kit (A24372), and Lipofectamine 3000 Reagent (L3000008) were from Life Technologies (Carlsbad, CA, United States); all- trans RA (R2625), crystal violet acetate (C5042), MTT (M5655), anhydrous DMSO (276855), DAPI (D9542), Akt activator SC79 (SML0749), and mTOR activator MHY1485 (SML0810) were from Sigma-Aldrich (St. Louis, MO, United States); PFA (15714) was from Electron Microscopy Sciences (Hatfield, PA, United States); recombinant human MANF (hMANF) (MANF-536H) was from Creative BioMart (New York, NY, United States); control CRISPR/Cas9 plasmid (sc-418922), mouse ARP double nickase plasmid (sc-428989-NIC), UltraCruz transfection reagent (sc-395739), plasmid transfection medium (sc-108062), and Akt inhibitor MK-2206 dihydrochloride (sc-364537) were from Santa Cruz Biotechnology (Dallas, TX, United States); Erk activator PDBu (12808), Erk inhibitor PD98059 (9900), and mTOR inhibitor Torin 1 (14379) were from Cell Signaling Technology (Beverly, MA, United States); pGEM-T-easy vector (A1360) was from Promega (Madison, WI, United States); 10-beta competent E. coli (C3019I) was from New England Biolabs (Ipswich, MA, United States); scrambled siRNA-GFP lentivector (LV015-G), Manf siRNA-GFP lentivector (279970940495), MANF-HA adenovirus (mouse) (279970540200), and CMV Null control adenovirus (000047A) were from Applied Biological Materials (Richmond, BC, Canada); PureLink Expi Endotoxin-free Maxi Plasmid Purification Kit (A31231) was from Thermo Fisher Scientific (Waltham, MA, United States); VECTASHIELD mounting medium (H-1400 and H-1500) was from Vector Laboratories (Burlingame, CA, United States); DC protein assay kit (5000112) was from Bio-Rad Laboratories (Hercules, CA, United States); Click-iT HPG Alexa Fluor Protein Synthesis Assay Kits (C10428) was from Invitrogen (Grand Island, NY, United States).. The following antibodies were used: anti-α-tubulin (T5168, Sigma-Aldrich); anti-ARMET/ARP (MANF) (ab67271 for C-terminus and ab67203 for N-terminus, Abcam, Cambridge, MA, United States); anti-HA-Tag (CST 3724), anti-phospho-Akt (Ser473) (CST9271), anti-Akt (CST9272), anti-phospho-Erk1/2 (CST 9101), anti-Erk1/2 antibody (CST 9102), anti-phospho-mTOR (Ser2448) (CST2971), anti-mTOR (CST2972), anti-phospho-p70 S6 (CST9204), anti-p70 S6 (CST2708), anti-Cas9 (CST 14697), and anti-β-Actin (CST3700) antibodies were all from Cell Signaling Technology; secondary antibodies conjugated to horseradish peroxidase (NA931V and NA934V) were from GE Healthcare Life Sciences (Pittsburgh, PA, United States); Alexa-488 conjugated anti-mouse (A21202), Alexa-594 conjugated anti-mouse (A11005) and Alexa-594 conjugated anti-rabbit antibodies (A11012) were from Life Technologies.

MANF overexpression facilitates RA-induced neurite outgrowth. (A) Representative images of N2a cells treated with DMSO or RA for 3 days with or without addition of recombinant hMANF (100 ng/ml). Cells were fixed and stained with crystal violet for visualization. (B) Average neurite length was measured, and analyzed by One-way ANOVA followed with the Tukey’s post hoc test, *** P < 0.0001 and n.s. not statistically significant compared to DMSO treated cells, ### P < 0.0001 compared to RA treated cells. The data were expressed as the mean ± SEM of three independent experiments. (C) Cells were lysed 48 h after adenovirus transduction and subjected to immunoblot to determine levels of MANF and HA tag expression. The size of the proteins (kDa) was labeled next to each band. (D) Endogenous and exogenous MANF protein levels were quantified and normalized to β-actin. The data were analyzed by Student’s t test, *** P < 0.0001. The data were expressed as the mean ± SEM of three independent experiments. (E) Representative images of AD-vector and AD-MANF transduced cells treated with RA for 24, 48, and 72 h. (F) Average neurite length of AD-vector and AD-MANF transduced cells after RA treatment was determined, and analyzed by Two-way ANOVA followed with the Bonferroni’s post hoc test, *** P < 0.001. The data were expressed as the mean ± SEM of three independent experiments.

MANF overexpression facilitates RA-induced neurite outgrowth. (A) Representative images of N2a cells treated with DMSO or RA for 3 days with or without addition of recombinant hMANF (100 ng/ml). Cells were fixed and stained with crystal violet for visualization. (B) Average neurite length was measured, and analyzed by One-way ANOVA followed with the Tukey’s post hoc test, *** P < 0.0001 and n.s. not statistically significant compared to DMSO treated cells, ### P < 0.0001 compared to RA treated cells. The data were expressed as the mean ± SEM of three independent experiments. (C) Cells were lysed 48 h after adenovirus transduction and subjected to immunoblot to determine levels of MANF and HA tag expression. The size of the proteins (kDa) was labeled next to each band. (D) Endogenous and exogenous MANF protein levels were quantified and normalized to β-actin. The data were analyzed by Student’s t test, *** P < 0.0001. The data were expressed as the mean ± SEM of three independent experiments. (E) Representative images of AD-vector and AD-MANF transduced cells treated with RA for 24, 48, and 72 h. (F) Average neurite length of AD-vector and AD-MANF transduced cells after RA treatment was determined, and analyzed by Two-way ANOVA followed with the Bonferroni’s post hoc test, *** P < 0.001. The data were expressed as the mean ± SEM of three independent experiments.

Addition of extracellular hMANF fails to rescue the neurite outgrowth defects in MANF KO cells. (A) Representative images of MANF KO N2a cells treated with DMSO or RA for 3 days with or without addition of recombinant hMANF (100 ng/ml). Cells were fixed and stained with crystal violet for visualization. (B) Average neurite length was measured, and analyzed by One-way ANOVA followed with the Tukey’s post hoc test, n.s. not statistically significant. The data were expressed as the mean ± SEM of three independent experiments.

Addition of extracellular hMANF fails to rescue the neurite outgrowth defects in MANF KO cells. (A) Representative images of MANF KO N2a cells treated with DMSO or RA for 3 days with or without addition of recombinant hMANF (100 ng/ml). Cells were fixed and stained with crystal violet for visualization. (B) Average neurite length was measured, and analyzed by One-way ANOVA followed with the Tukey’s post hoc test, n.s. not statistically significant. The data were expressed as the mean ± SEM of three independent experiments.

Pharmacological inhibition of Akt, Erk, and mTOR blocks MANF-enhanced neurite outgrowth in response to RA. (A) Representative images of N2a cells treated with DMSO or RA or RA+inhibitors for 3 days with or without addition of recombinant hMANF (100 ng/ml). Cells were fixed and stained with crystal violet for visualization. (B) Average neurite length was measured, and analyzed by One-way ANOVA followed with the Tukey’s post hoc test, *** P < 0.0001 compared to DMSO treated cells, ### P < 0.0001 compared to RA treated cells. The data were expressed as the mean ± SEM of three independent experiments.

Pharmacological inhibition of Akt, Erk, and mTOR blocks MANF-enhanced neurite outgrowth in response to RA. (A) Representative images of N2a cells treated with DMSO or RA or RA+inhibitors for 3 days with or without addition of recombinant hMANF (100 ng/ml). Cells were fixed and stained with crystal violet for visualization. (B) Average neurite length was measured, and analyzed by One-way ANOVA followed with the Tukey’s post hoc test, *** P < 0.0001 compared to DMSO treated cells, ### P < 0.0001 compared to RA treated cells. The data were expressed as the mean ± SEM of three independent experiments.

Publication :
MANF protects pancreatic acinar cells against alcohol-induced endoplasmic reticulum stress and cellular injury (2021)
MANF inhibits Sjo ̈gren’s syndrome salivary gland epithelial cell apoptosis and antigen expression of Ro52/SSA through endoplasmic reticulum stress/ autophagy pathway (2023)

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