Antibody data
- Antibody Data
- Antigen structure
- References [0]
- Comments [0]
- Validations
- Western blot [1]
- Immunocytochemistry [3]
- Immunohistochemistry [3]
- Flow cytometry [2]
- Other assay [3]
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- Product number
- MA3-917 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Calmodulin Monoclonal Antibody (2D1)
- Antibody type
- Monoclonal
- Antigen
- Purifed from natural sources
- Description
- MA3-917 detects calmodulin from human, bovine, chicken, Chlamydomonas, Dictyostelium, mouse and rat samples. This antibody does not detect parvalbumin, tropinin, S-100, or myosin light chain kinase (MLCK). MA3-917 has been successfully used in Western blot, immunocytochemistry and ELISA procedures. By Western blot, this antibody detects a 17 kDa protein representing calmodulin from Dictyostelium cell lysate. Addition of EGTA to buffers completely inhibits antibody staining. Immunohistochemical staining of calmodulin in Dictyostelium cells with MA3-917 results in staining of the contractile vacuoles. The MA3-917 antigen is calmodulin purified from Dictostelium discoideum.
- Reactivity
- Human, Mouse, Rat, Bacteria, Bovine, Chicken/Avian
- Host
- Mouse
- Isotype
- IgG
- Antibody clone number
- 2D1
- Vial size
- 200 µL
- Concentration
- 1 mg/mL
- Storage
- -20° C, Avoid Freeze/Thaw Cycles
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot of purified calmodulin using Product # MA3-917 and Product # MA3-918.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
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- Experimental details
- Immunofluorescent analysis of Calmodulin using Calmodulin Monoclonal antibody (2D1) (Product # MA3-917) shows staining in A2058 melanoma cells. Calmodulin 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 Calmodulin (Product # MA3-917) at a dilution of 1:20 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.
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- Invitrogen Antibodies (provider)
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- Experimental details
- Immunofluorescent analysis of Calmodulin using Calmodulin Monoclonal antibody (2D1) (Product # MA3-917) shows staining in C6 glioma cells. Calmodulin 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 Calmodulin (Product # MA3-917) at a dilution of 1:20 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.
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- Invitrogen Antibodies (provider)
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- Experimental details
- Immunofluorescent analysis of Calmodulin using Calmodulin Monoclonal antibody (2D1) (Product # MA3-917) shows staining in HeLa cells. Calmodulin 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 Calmodulin (Product # MA3-917) at a dilution of 1:20 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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- Invitrogen Antibodies (provider)
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- Experimental details
- Immunohistochemistry was performed on normal biopsies of deparaffinized Rat cerebellum 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 1:20 with a mouse monoclonal antibody recognizing Calmodulin (Product # MA3-917) 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.
- Submitted by
- Invitrogen Antibodies (provider)
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- Experimental details
- Immunolocalization of calmodulin in rat brain using Product # MA3-917.
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- Invitrogen Antibodies (provider)
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- Experimental details
- Immunohistochemistry was performed on normal biopsies of deparaffinized Rat testis 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 1:20 with a mouse monoclonal antibody recognizing Calmodulin (Product # MA3-917) 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
- Flow cytometry analysis of Calmodulin in MCF-7 cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were penetrated by dropping the supernatant, adding 90% methanol and incubated for 10 minutes at room temperature. Follwing penetration, cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a Calmodulin monoclonal antibody (Product # MA3-917) at a dilution of 2 µg/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.
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- Flow cytometry analysis of Calmodulin in PC12 cells compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were penetrated by dropping the supernatant, adding 90% methanol and incubated for 10 minutes at room temperature. Follwing penetration, cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a Calmodulin monoclonal antibody (Product # MA3-917) at a dilution of 2 µg/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.
Supportive validation
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- Figure 8 Relocalization of CaM in NCKX4 transfected HEK293 cells. HEK293 cells grown on coverslips were transfected with cDNA constructs, as indicated for each row at right: vector-only; NCKX4; NCKX4 and the Ca 2+ binding-defective CaM mutant (CaM Mut); NCKX4 I328D/F334D double mutant (NCKX4 Mut). Fixed and permeabilized cells were subsequently stained with a combination of either: A , mouse anti-CaM and rabbit PSD anti-NCKX4 antibodies, Ms CaM Ab and Rb NCKX4 Ab, respectively; B , rabbit anti-CaM and mouse N4MAb anti-NCKX4 antibodies, Rb CaM Ab and Ms NCKX4 Ab, respectively, as noted above each panel. DAPI stain is shown to visualize the nuclei of all the cells within the field of view. All photographs taken for cells stained with the same antibody were captured using identical exposure settings. Contrast and brightness adjustments made in Adobe Photoshop were applied identically to all images for the same antibody. The merged images were further adjusted so overlap (seen in yellow ) between the staining patterns could be appreciated more readily. Scale bar (top left panel) corresponds to 25 mum. The insets at top right of the merged image panels correspond to a fourfold magnified view of one cell from the field of view. The images in this figure are representative of at least three independent experiments.
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- Figure 5 Expression level of skeletal muscle proteins, the amount of releasable Ca 2+ from the SR to the cytosol, and the resting cytosolic Ca 2+ level in primary skeletal myotubes. ( a ) The immunoblot analysis of proteins mediating Ca 2+ movements and handling in skeletal muscle was conducted using the lysate of myotubes treated with sildenafil. Fifteen proteins were examined, and no expression levels were changed by sildenafil (the expression level of proteins is presented as bar graphs in Supplementary Figure 5 ). alpha-Actin was used as a loading control. At least three independent experiments per protein were conducted and a representative result is presented. JP, junctophilin; CSQ, calsequestrin; Mg, mitsugumin; CaM, calmodulin. The amount of releasable Ca 2+ from the SR to the cytosol in response to TG (2.5 mu M ) ( b ) or resting cytosolic Ca 2+ level ( c ) was examined in myotubes treated with sildenafil, and histograms are shown for the normalized peak amplitude or area under the peak to the mean value of those from untreated controls as described in the 'Materials and methods' section. TG was applied to myotubes in the absence of extracellular Ca 2+ to avoid extracellular Ca 2+ entry. The results are presented as the means+-s.e. for the number of experiments presented in the parentheses of Table 2 . *, and the significant difference was compared with the untreated control ( P
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- Fig 3 Decreased NO concentrations and impairment of eNOS localization in EHD2 del/del mesenteric arteries. (A) NO abundance was determined by DAF staining of EHD2 del/+ and del/del mesenteric arteries either untreated or pre-treated with L-NAME. (B) DAF staining intensity is significantly reduced for EHD2 del/del arteries compared to EHD2 del/+ (L-NAME: n(del/+) = 51/3; n(del/del) = 43/3; untreated: n(del/+) = 121/3; n(del/del) = 127/3). (C) STED imaging of eNOS (blue) and Cav1 (magenta) in EHD2 del/+ mesenteric artery cryostat sections illustrates eNOS localization at the plasma membrane of the small arteries in close proximity to caveolae (merge). (D) EHD2 del/del mesenteric arteries showed reduced eNOS staining at the plasma membrane, instead eNOS was detected within the cytosol of endothelial cells. Scale bar 4 mum. See also S4 Fig . (E) Plasma membrane eNOS staining was analyzed and revealed decreased eNOS fluorescence intensity in EHD2 del/del mesenteric arteries compared to EHD2 del/+ (n(del/+) = 83/3; n(del/del) = 81/3). (F) Analysis of total eNOS protein concentration in tissue lysates obtained from EHD2 del/+ and del/del small vessels by Western Blot. Calmodulin was used as loading control. Graphs illustrate each replicate with mean +/- SE, t-test or Mann Whitney U test were used to calculate significance, *** P