MA3-940

antibody from Invitrogen Antibodies

Targeting: CAPN1 CANP, CANPL1, muCANP, muCL

Provider product page for MA3-940

Western blot

Immunocytochemistry

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [52]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [3]
Immunohistochemistry [2]
Other assay [4]

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Product number: MA3-940 - Provider product page
Provider: Invitrogen Antibodies
Product name: Mu-Calpain Monoclonal Antibody (9A4H8D3)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: MA3-940 detects mu-calpain from human platelets and erythrocytes, bovine platelets, heart and skeletal muscle, rat myoblasts, kidney, liver and spleen, mouse lung, pig cultured cells and hamster and rabbit samples. This antibody does not cross-react with m-calpain, n-calpain, calmodulin or calpastatin. MA3-940 has been successfully used in Western blot, immunofluorescence, immunohistochemistry, and immunocytochemistry procedures. By Western blot, this antibody detects an 80 kDa protein representing mu-calpain from human platelets and erythrocytes and HeLa and A431 cell lysates. Immunocytochemical staining of mu-calpain in LLC-PK1 cells with MA3-940 results in diffuse cytoplasmic staining. This product has not been shown to be effective in immunoprecipitation experiments. The MA3-940 antigen is purified bovine skeletal muscle 80 kDa mu-calpain subunit. This antibody recognizes an epitope between amino acids 465-520 (domain III) of human mu-calpain.
Reactivity: Human, Mouse, Rat, Bovine, Hamster, Porcine, Rabbit
Host: Mouse
Isotype: IgG
Antibody clone number: 9A4H8D3
Vial size: 100 µg
Concentration: 1.0 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

CAPN1 protein structure - MA3-940 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of HeLa (Lane 1), A-431 (Lane 2), MOLT-4 (Lane 3), SK-OV-3 (Lane 4), LNCaP (Lane 5) and PC-3 (Lane 6). The blot was probed with Mouse Anti-Mu-Calpain Monoclonal Antibody (Product # MA3-940, 2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). An 80 kDa band corresponding to Mu-Calpain was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of Mu-Calpain was achieved by transfecting HeLa cells with hnRNP K specific validated siRNAs (Silencer® select Product # s491). Western blot analysis (Fig. 1) was performed using whole cell extracts from the Mu-Calpain knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blots were probed with Anti-Mu-Calpain Monoclonal Antibody (Product # MA3-940, 1 µg/mL) and Goat Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. 2). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Mu-Calpain.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Mu-Calpain in HeLa Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a Mu-Calpain monoclonal antibody (Product # MA3-940) at a dilution of 1:100 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503). Mu-Calpain staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification. This image was taken using unpurified ascites antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Mu-Calpain in U251 Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a Mu-Calpain monoclonal antibody (Product # MA3-940) at a dilution of 1:20 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503). Mu-Calpain staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification. This image was taken using unpurified ascites antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Mu-Calpain in MCF-7 Cells. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with a Mu-Calpain monoclonal antibody (Product # MA3-940) at a dilution of 1:20 overnight at 4 C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35503). Mu-Calpain staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Images were taken at 60X magnification. This image was taken using unpurified ascites antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on normal biopsies of deparaffinized human kidney 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 Mu-Calpain (Product # MA3-940) 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. This image was taken using unpurified ascites antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry was performed on normal biopsies of 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 1:20 with a Mouse Monoclonal Antibody recognizing Mu-Calpain (Product # MA3-940) 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. This image was taken using unpurified ascites antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2. ARL-13/Arl13b associates with ciliary membranes via palmitoyl anchors. (A) N-terminal Pal motif ARL-13 variants (delPal and rPal) fail to localize to amphid (head) and phasmid (tail) cilia (c) and diffusely mislocalize in cell bodies (cb) and dendrites (d). C-terminal deletion ARL-13(del203-370) is also mislocalized, but membrane associations are maintained (arrows). Insets show high magnification images of PHA/B cilia. (B) HsArl13b ciliary localization requires an N-terminal Pal motif. Ciliated RPE1 cells transfected with GFP-tagged Arl13b(WT) or a C8S/C9S variant and costained for ciliary axonemes using acetylated alpha-tubulin antibody are shown. Merged images show that Arl13b(C8S/C9S) is highly diffuse and nonmembrane associated, with weak signals in cilia. The boxed regions are shown at high magnification in the insets. Dashed lines outline the cell. (C) Loss of Pal motif shifts HsArl13b to cytosolic fractions. A subcellular fractionation scheme for Flag-tagged Arl13b (WT and variants) transiently expressed in 293T cells is shown. Supernatants (Sup.) and pellets (Pell) were probed for Arl13b::Flag using Western blotting and an anti-Flag antibody. Calpain and Na + /K + ATPase proteins mark cytosol and membrane fractions, respectively. Arl13b(WT) and Arl13b(1-357) pellet with Na + /K + ATPase membrane proteins after 20,000 g spins, whereas Arl13b(C8S/C9S) remains in calpain-enriched cytosolic supernatants even after 200,000 g spins. (D) Arl13b is palmitoylated in

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 1 (a) A representative set of western blot images showing 1 day vs. 7 days postmortem change of desmin, troponin T, calpain-1, HSP27, alphabeta-crystallin and cytochrome c of porcine LD ( longissimus dorsi ) and PM ( psoas major ) muscles with two production replicates. Reference: consistently use a 45 min postmortem LD for calpain-1 and a 7 days postmortem LD for desmin, troponin T, HSP27, alphabeta-crystallin and cytochrome c. July-Jan: Pigs weaned in July 2016 and harvested in January 2017; Apr-Sep: Pigs weaned in April 2017 and harvested in September 2017. All western blots were performed in separate gels in independent manners (please see supplementary information for the entire western blot images). (b-l) Densitometry analysis of each protein bands.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: EV3 Figure PI(3)P loss inhibits SV endocytosis via calpain 2 activation A Levels of various synaptic proteins in DMSO (0.1%) and VPS34IN1 (10 uM) treated cultured cerebellar granule neurons analyzed by western blot. B Surface retention of Synaptophysin-pHluorin 90 s poststimulus is plotted for each of the four successive stimulation trains in neurons treated with VPS34IN1 (10 uM) and Calpeptin (10 uM). Mean +- SEM; >= 9 images per condition from 3 independent experiments; *** P < 0.001; **** P < 0.0001; Two-way ANOVA. C Processing of p35 to p25 in cultured hippocampal neurons treated with VPS34IN1 (10 uM) for 1 h, analyzed by western blot. Cleavage of p35 to p25 was reduced in neurons simultaneously treated with ALLN (100 uM) but not with Roscovitine (Ros, 10 uM). D, E Cultured hippocampal neurons were cotransfected with an mRFP expression plasmid and shCtrl, shCAPN1, or shCAPN2 and stained for Calpain 1 in D or Calpain 2 in E. Scale bar, 10 um. The relative mean fluorescence intensity is plotted. >= 8 images per condition; **** P < 0.0001; Student's t -test. Source data are available online for this figure.