MA3-910

antibody from Invitrogen Antibodies

Targeting: ATP2A2 ATP2B, DAR, SERCA2

Provider product page for MA3-910

Western blot

Immunocytochemistry

Immunohistochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [168]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [4]
Immunohistochemistry [3]
Other assay [25]

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Product number: MA3-910 - Provider product page
Provider: Invitrogen Antibodies
Product name: SERCA2 ATPase Monoclonal Antibody (IID8)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: MA3-910 detects sarcoplasmic or endoplasmic reticulum calcium 2 (SERCA2) ATPase from bovine, canine, human, pig, rabbit and rat tissues. This antibody does not detect frog SERCA2 ATPase (J. Biol. Chem., Feb 2001; 276: 3911 - 3919). MA3-910 detects SERCA2 ATPase and does not discriminate between the two isoforms. MA3-910 has been successfully used in Western blot, immunofluorescence, immunocytochemistry, and immunohistochemistry. By Western blot, this antibody detects an ~110 kDa protein representing SERCA2 ATPase from canine skeletal muscle triad preparations. Immunofluorescence staining of SECRA2 ATPase in rabbit skeletal muscle with MA3-910 results in strong labeling of the entire type I (slow) myofiber consistent with sarcoplasmic reticulum localization. MA3-910 is not recommended for Western blot detection of rat SERCA2. The MA3-910 antigen is purified canine cardiac sarcoplasmic reticulum.
Reactivity: Human, Mouse, Rat, Bovine, Canine, Porcine, Rabbit
Host: Mouse
Isotype: IgG
Antibody clone number: IID8
Vial size: 100 µL
Concentration: 1 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

ATP2A2 protein structure - MA3-910 shown in red.

Supportive validation

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Experimental details: Western blot analysis of SERCA2 ATPase was performed by loading 25 µg of rat skeletal muscle (lane 1) and human skeletal muscle (lane 2) onto an SDS polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked at 4ºC overnight. The membrane was probed with a SERCA2 ATPase monoclonal antibody (Product # MA3-910) at a dilution of 1:1000 (rat muscle) and 1:2000 (human muscle) overnight at 4°C, washed in TBST, and probed with an HRP-conjugated secondary antibody for 1 hr at room temperature in the dark. Chemiluminescent detection was performed using Pierce ECL Plus Western Blotting Substrate (Product # 32132). Results show a band at ~110 kDa.

Supportive validation

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Experimental details: Western blot was performed using Anti-SERCA2 ATPase Monoclonal Antibody (IID8) (Product # MA3-910) and bands at ~114, 109 kDa corresponding to SERCA2 ATPase was observed across the cell lines tested. Whole cell extracts (30 µg lysate) of A549 (Lane 1), T-47D (Lane 2), PANC-1 (Lane 3), C2C12 (Lane 4), HeLa (Lane 5), MDCK (Lane 6), RD (Lane 7) and Reh (Lane 8) were electrophoresed using Novex® NuPAGE® 4-12% % Bis-Tris gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005)..

Supportive validation

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Experimental details: Immunofluorescent analysis of SERCA2 ATPase using SERCA2 ATPase Monoclonal antibody (IID8) (Product # MA3-910) shows staining in A549 cells. SERCA2 ATPase staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing SERCA2 ATPase (Product # MA3-910) at a dilution of 1:100-1:200 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

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Experimental details: Immunofluorescent analysis of SERCA2 ATPase using SERCA2 ATPase Monoclonal antibody (IID8) (Product # MA3-910) shows staining in HeLa cells. SERCA2 ATPase staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing SERCA2 ATPase (Product # MA3-910) at a dilution of 1:100-1:200 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of SERCA2 ATPase using SERCA2 ATPase Monoclonal antibody (IID8) (Product # MA3-910) shows staining in U251 glioma cells. SERCA2 ATPase staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing SERCA2 ATPase (Product # MA3-910) at a dilution of 1:100-1:200 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of SERCA2 was performed using 70% confluent log phase MDCK cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with SERCA2 ATPase Monoclonal Antibody (Product # MA3-910) at 5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415). Panel d represents the merged image showing Cytoplasmic (mostly ER) localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

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Experimental details: Immunohistochemistry was performed on normal deparaffinized human Heart tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a mouse monoclonal antibody recognizing SERCA2 ATPase (Product # MA3-910) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on normal deparaffinized human Tonsil tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a mouse monoclonal antibody recognizing SERCA2 ATPase (Product # MA3-910) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on normal deparaffinized human Liver tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:200 with a mouse monoclonal antibody recognizing SERCA2 ATPase (Product # MA3-910) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

Supportive validation

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Experimental details: Fig. 4 Changes of ATP2A/SERCA2a and HSPA2 at the mRNA and protein levels in HCM versus control hearts. a A plot of the joint component loadings of RNA-seq data showed ATP2A2 and HSPA2 were two major players in discriminating HCM hearts from controls. The dashed lines on both positive and negative sides indicate the cutoff threshold, with genes with a large contribution to the joint component falling outside of the dash lines. b ATP2A2 mRNA and protein levels in HCM and control samples at the mRNA and protein levels. c HSPA2 mRNA and protein levels expression in HCM and control samples. **: P < 0.01, ***: P < 0.001, ****: P < 0.0001. d Representative immunohistochemistry staining showing higher HSPA2 staining intensity in HCM heart as compared to control. Scale bar = 400 um (control sample) and 800 um (HCM sample). e Representative immunofluorescence staining showing HSPA2 aggregates in an HCM heart (indicated by the arrow), whereas the control shows diffuse staining of the cytoplasm without aggregates. WGA-AF488 appears in gray to visualize the cell membrane, and DAPI appears in blue to visualize the nuclei. Scale bar = 16 um

Supportive validation

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Experimental details: Fig. 6 Immunofluorescence (IF) staining of selected candidates in control and HCM hiPSC-cardiomyocytes. The same amount of control and HCM hiPSC-cardiomyocytes (20,000 cells per well) were seeded to the plate and cultured for 27 days prior to the staining. a Representative IF staining showing the expression of KLF15 in HCM and control cardiomyocytes (KLF15: red; ACTN2: green; blue: cell nuclei). b Representative IF staining showing disrupted ACTN2 in HCM cardiomyocytes compared to controls (ACTN2: green; blue: cell nuclei). c Representative IF staining showing suppressed ATP2A2 in HCM cardiomyocytes compared to controls (ATP2A2: red; blue: cell nuclei). Nuclei were stained by Hoechst. All overview images were taken at 20 x magnification, scale bar = 100 um. All zoomed regions were taken under Zoom Factor = 1.5

Supportive validation

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Experimental details: Figure 7 Variations of gene and protein expression involved in Ca 2+ handling in response to 10-day bed rest Calsequestrin1-2, SERCA1, SERCA2 and TOM20 amount in muscle homogenates as obtained by western blot at baseline (BR0) and after 10 days of bed rest (BR10) ( A ). Representative western blot of CASQ1-2, SERCA1, SERCA2 and TOM20 ( B ). CASQ1 and CASQ2 gene expression (encoding for Calsequestrin1 and Calsequestrin2, respectively) ( C , D ), ATP2A1 and ATP2A2 gene expression (encoding for SERCA1 and SERCA2, respectively) ( E , F ); RYR1 gene expression (encoding for Ryanodine Receptor 1) ( G ). All RNA transcripts are reported as normalized read count at BR0, after 5 days of bed rest (BR5) and at BR10. Results shown as means +- SD, individual data represented as scatter plots. * P < 0.05 BR10 vs BR0, ** P < 0.01 BR10 vs BR0, **** P < 0.0001 BR10 vs BR0.

Supportive validation

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Experimental details: Nnat increases ER calcium storage by facilitating SERCA function. ( A and B ) Left: cortical neurons were transfected with ER-GCaMP6 or GCaMP6s to measure ER or cytosolic level. Examples of images before and after application of 50 muM IO. Right: quantification of calcium concentration of ER or cytoplasm at rest. Scale bars: 10 mum. n = approximately 42-45 for each group. ( C and D ) Coimmunoprecipitation results showed Nnat could interact with SERCA2 but not IP3R. n = 3 independent experiments. ( E ) Upper left panel: cdn1163 activated SERCA and promoted Ca 2+ influx to ER. Upper right and lower panels: representative time courses of cdn1163 Ca 2+ imaging using GCaMP6s indicator in WT neurons infected with Nnat lentivirus. Individual cell trace (gray) and average trace (black) are shown for each group. ( F ) Upper left panel: CPA inhibited SERCA and promoted Ca 2+ efflux from ER. Upper right and lower panels: representative time courses of CPA Ca 2+ imaging using GCaMP6s indicator in WT neurons infected with Nnat lentivirus. Individual cell trace (gray) and average trace are shown for each group. ( G and I ) Quantification of peak values normalized to control group of cdn1163 Ca 2+ imaging and CPA Ca 2+ imaging. n = approximately 32-38 neurons for cdn1163 Ca 2+ imaging and n = approximately 40-43 neurons for CPA Ca 2+ imaging. ( H and J) Quantification of slope in cdn1163 Ca 2+ imaging and CPA Ca 2+ imaging. n = approximately 43-51 neurons for cdn1163 Ca 2+ imaging and n = a

Supportive validation

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Experimental details: Figure 2. A, SERCA-2 mRNA levels assessed from Northern blots decreased within 4 days after initiation of cholesterol feeding and fell to &ap30&percnt from days 8 through 70. B, SERCA-2 protein levels assessed from Western blots remaining unchanged over first 16 days on cholesterol diet and fell &ap55&percnt by day 70. n&equals3 animals per time point. C, Time-dependent SERCA-2 activity assessed with a TSG-sensitive 45 Ca uptake technique into LV SR was decreased &ap50&percnt after 70 days on high-cholesterol diet. n&equals3 control and 3 diet animals Differences between time points were tested with ANOVA with a post hoc Bonferroni correction. Data are mean+-SEM; P

Supportive validation

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Experimental details: Figure 4 Immunolocalization of myospryn and minispryn in isolated cardiomyocytes. Enzymatically isolated guinea pig ventricular cardiomyocytes were fixed and processed for confocal immunofluorescence microscopy using the indicated antibodies. ( a - c ) At low magnification myospryn (a) immunoreactivity is visible in discontinuous striations that run perpendicular to the long axis of the cardiomyocyte partially overlapping the nucleus (arrow in a). ( d - f ) Higher magnification images show that myospryn immunoreactivity is present in distinct punctae that co-localise with ryanodine receptors (e) in the SR (f and inset). ( g - i ) Anti-minispryn (g) immunoreactivity also appears in discontinuous striations running the length of the cardiomyocyte and is also present in the nucleus (arrow in g). ( j - l ) In common with myospryn, higher magnification images show that minispryn partially co-localises with ryanodine receptors (k) in the SR (l and inset). ( m-o ) Myospryn immunoreactivity overlaps the Z-line labelled with an anti-alpha-actinin antibody (m, n, o). ( p - r ) Similarly, minispryn immunoreactivity also partially overlaps alpha-actinin at the Z-line (p, q, r). ( s - x ) Cardiomyocytes labelled with either anti-SERCA2 (s, t, u) or anti-sarcalumenin (v, w, x) antibodies and myospryn antibodies demonstrate that myospryn immunoreactivity (arrowheads in s) appears to be restricted to the j-SR rather than the l-SR (arrows in t).

Supportive validation

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Experimental details: Cytoskeletal reorganization does not alter calcium-channel-induced Ca 2+ release in arterial myocytes. ( A ) Example of depolarization (70 mM K + ) and ATP (100 muM) induced Ca 2+ release in a myocyte and confocal fluorescent staining of the cell with markers of the SR (antibodies against Ca ATPase, red) and cytoskeletal microfilaments (antibody against actin, green). ( B ), ( C ) Similar experiments as in (A) performed in cells subjected to prior incubation with calyculin A [(B) 100 nM, 30 min, n = 8 experiments] or jasplakinolide [(C) 1 muM, 120 min, n = 7 experiments]. Note in (B) and (C) the actin ring separating the plasma membrane and the cytosolic organella.