MA5-12272

antibody from Invitrogen Antibodies

Targeting: SNCA NACP, PARK1, PARK4, PD1

Provider product page for MA5-12272

Western blot

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [19]
Comments [0]
Validations
Immunocytochemistry [3]
Other assay [8]

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Product number: MA5-12272 - Provider product page
Provider: Invitrogen Antibodies
Product name: alpha Synuclein Monoclonal Antibody (Syn 211)
Antibody type: Monoclonal
Antigen: Recombinant full-length protein
Description: MA5-12272 targets Synuclein in IHC (P), ICC/IF, IP, and WB applications and shows reactivity with Human and Rat samples. The MA5-12272 immunogen is human recombinant alpha-synuclein.
Reactivity: Human, Rat
Host: Mouse
Isotype: IgG
Antibody clone number: Syn 211
Vial size: 500 µL
Concentration: 0.2 mg/mL
Storage: 4° C

SNCA protein structure - MA5-12272 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Synuclein (green) showing staining in the cytoplasm of PC12 cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Synuclein monoclonal antibody (Product # MA5-12272) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Synuclein (green) showing staining in the cytoplasm and membrane of U251 cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Synuclein monoclonal antibody (Product # MA5-12272) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Synuclein (green) showing staining in the cytoplasm and membrane of U87-MG cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Synuclein monoclonal antibody (Product # MA5-12272) in 3% BSA-PBS at a dilution of 1:100 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 Aggregates Are Comprised of Cytoskeletal Proteins (A) LRRK2 G2019S aggregates were positive (indicated by arrows) for betaIII tubulin (green) and peripherin (red), tau (green), MAP2 (red), and LRRK2 (green). Aggregates were generally negative (indicated by arrowheads) for SNCA and ubiquitin (green). (B-D) LRRK2 G2019S sensory neuron cultures display significantly increased levels of (B and C) tau and (B and D) phospho-tau by western blot and densitometry. * p < 0.01 by ANOVA; n = 6 independent experiments. The scale bar represents 50 mum. See also Figure S3 .

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 Alpha-synuclein perivascular immunoreactivity in postmortem sections from sporadic patients with PD. For these experiments, sections from three patients with PD (PD16, disease duration 18 years; PD45, disease duration 19 years; and PD121, disease duration 4 years) and three healthy controls (CO28, CO34, and CO48), kindly supplied by the Parkinson's UK Brain Bank, were analyzed. Briefly, sections were treated for antigen retrieval with 10 mM sodium citrate (20' at 95degC) and 10% formic acid (15' at RT). After 1 h incubation at room temperature (RT) with blocking solution (2% w/vol bovine serum albumin, 3% vol/vol normal goat serum, 0.3% Triton X-100 diluted in PBS 0.1 M pH 7.4) the 5-mum slices were subjected to either single alpha-synuclein (Sin211 MA5-12272 Thermo Fisher Scientific, Waltham, USA; dilution 1:500) or double laminin alpha2 (4H8-2, abcam ab11576; dilution 1:100)/alpha-synuclein (Sin211, MA5-12272 Thermo Fisher; dilution 1:500) immunolabeling according to previously described protocols (, ). Single alpha-synuclein immunopositive signal was revealed by Blue Alkaline Phosphatase (Vector Laboratories, Burlingame, CA) acquired by using a 40X objective, while for double immunolabeling laminin alpha2 was revealed by brown 3,3-diaminobenzidine (DAB) and alpha-synuclein by violet (Nickel supplemented) DAB (Vector laboratories) and acquired by a 100X objective. All the images were acquired by using an inverted light microscope (Olympus BX41; Olympus, Milan, Ital

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 3 Mn-dependent enhanced alpha-Syn expression disturbs the phosphorylation of NMDA receptors in vivo and in vitro. a , b After treatment with Mn, western blotting was used to measure the level of alpha-Syn overexpression in the hippocampus. c - f The levels of phosphorylated NMDA receptors (GluN2B and GluN2A) were measured by western blotting in vivo. n = 6. g , h HT22 cells were transfected with LV-alpha-Syn shRNA, and the cytotoxicity was measured using the CCK-8 assay and lactate dehydrogenase (LDH) release assay. i - m The levels of phosphorylated NMDA receptors (GluN2B and GluN2A) and alpha-Syn expression were measured by western blotting in vitro. n = 4. ** P < 0.01 compared to their control counterparts; # # P < 0.01, and # P < 0.05 for comparison between the Mn treatment group.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4 Brain-derived neurotrophic factor (BDNF) interfered with the interaction of alpha-Syn and TrkB. a , b After pretreatment with BDNF and Mn in HT22 cells, representative images of PLA show the interaction between alpha-Syn and TrkB (red spots), and the nucleus was stained in blue (DAPI). Scale bars = 50 mum. c , d The immunoprecipitation analysis of alpha-Syn and TrkB is shown. e - g After treating the mice with Mn, the levels of BDNF and TrkB were evaluated by western blotting. n = 6. h - j For HT22 cells transfected with the LV-alpha-Syn shRNA, the levels of BDNF and TrkB were evaluated by western blotting after Mn treatment. k - m After pretreatment with BDNF and Mn in HT22 cells, the levels of TrkB and alpha-Syn expression were evaluated by western blotting. n = 4. ** P < 0.01, and * P < 0.05 compared to their control counterparts; ## P < 0.01, and # P < 0.05 for comparison between the Mn treatment group.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Ang-(1-7) attenuates alpha-synuclein accumulation in the rotenone-induced cell model in a MasR-dependent manner . (A) The expression of alpha-syn in four groups was examined by Western blot assay. (B) Quantitative results of alpha-syn expression. (C) Cells were marked by an anti-alpha-syn antibody (red), nuclei were counterstained with DAPI (blue), and immunofluorescence was observed by fluorescent microscopy (original magnification, 630x). Expression of alpha-syn was higher in the ROT group and was downregulated when incubated with Ang-(1-7). However, the influence caused by Ang-(1-7) was completely abolished with A-779 co-treatment. (D) Mean alpha-syn fluorescence intensity (ratio to control) of four groups. Data are expressed as the mean the +- SD ( n = 3; one-way analysis of variance followed by Tukey's post hoc test). # P < 0.05, ## P < 0.01, vs . control group; + P < 0.05, ++ P < 0.01, vs . ROT group; * P < 0.05, ** P < 0.01, vs . ROT + Ang-(1-7) group. Ang-(1-7): Angiotensin-(1-7); DAPI: 4',6-diamidino-2-phenylindole; MasR: Mas receptor; ROT: rotenone; alpha-syn: alpha-synuclein.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6 Activation of autophagy contributes to the clearance of alpha-synuclein accumulation in the rotenone-induced cell model . (A)The p62 and LC3-II levels were determined by Western blot assay. (B) Quantitative results of p62 expression. (C) Quantitative results of LC3-II expression. (D) The expression of alpha-syn was detected by Western blot assay. (E) Quantitative results of alpha-syn expression. (F) The number of LAMP2A-positive cells (green) and alpha-syn-positive cells (red) by immunofluorescence were observed with a fluorescent microscope (original magnification, 630x). (G) Mean LAMP2A fluorescent intensity (ratio to control) of four groups. (H) Mean alpha-syn fluorescence intensity (ratio to control) of four groups. Data are expressed as the mean +- SD (B, C, E; n = 3; one-way analysis of variance followed by Tukey's post hoc test). ## P < 0.01, ### P < 0.001, #### P < 0.0001, vs . control group; + P < 0.05, ++ P < 0.01, +++ P < 0.001, ++++ P < 0.0001, vs . ROT group; * P < 0.05, *** P < 0.001, **** P < 0.0001, vs . ROT + Ang-(1-7) group. 3-MA: 3-Methyladenine; Ang-(1-7): angiotensin-(1-7); DAPI: 4',6-diamidino-2-phenylindole; LAMP2A: lysosomal-associated membrane protein 2A; LC3: microtubule associated protein 1 light chain 3; ROT: rotenone; alpha-syn: alpha-synuclein.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 Dopamine transporter (DAT) staining in the caudate putamen of PD patients and age-matched controls. ( A ) Representative photomicrograph showing the negative control produced by performing DAT immunolabeling on cerebellum sections. Please note the absence of staining that is indicative of the specificity of the DAT antibody used for this study; ( B , D , E , G , H , G1 , H1 , G2 , G3 ) Representative images showing DAT immunolabeling in the caudate putamen of PD patients and age-matched controls subjects. DAT immunolabeling showed a small dot-like appearance with widespread distribution in the control sections ( B , D , E ). Please note that in spite of the overall decrease of immunolabeling, in the PD brains, the DAT signal was accumulated in big neuropil-like dots ( G , H , G1 , H1 , G2 , arrowheads) and in few neuritic-like structures ( G , G1 , G3 , arrows); ( C , F , I , I1 , L ) Panels are showing alpha-synuclein immunolabeling in the caudate putamen of PD patients and age-matched control subjects. Alpha-synuclein immunolabeling showed a widespread distribution in the grey matter in control sections ( C , F ). Please note the presence of neuropil-like alpha-synuclein-positive dots ( I , I1 , L , arrowheads) and LB-like structures in the PD brains ( L , arrow). Scale bars: ( A - C , G - I ) 100 um, ( D - F , G1 , H1 , I1 ) 50 um, ( G2 , G3 , L ) 20 um.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 Dopamine transporter (DAT)/alpha-synuclein in situ PLA performed on the caudate putamen of PD patients and control subjects. ( A - C ) Panels showing the negative control, performed by omitting the DAT antibody, of the in situ PLA assayed on the caudate putamen of a control subject. The absence of the PLA-positive signal is indicative of the specificity of the assay; ( D - U ) Representative images showing DAT/alpha-synuclein in situ PLA performed on the caudate putamen of PD patients ( J - U ) and age-matched-controls (control, D - I ). The presence of a PLA positivity as a red fluorescent signal is indicative of the interaction between the two proteins. Please note the marked redistribution of the PLA-positivity in the caudate putamen of PD samples ( L , O , R ) when compared to the control subjects ( F , I ). In particular, in the PD brains, the PLA signal was particularly abundant within big ( O , arrowheads) and small ( L , R , arrowheads) clumps or neuritic-like structures ( U ). Scale bar: 20 mum.