702736

antibody from Invitrogen Antibodies

Targeting: EMC3 TMEM111

Antibody data

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Validation data

Product number: 702736 - Provider product page
Provider: Invitrogen Antibodies
Product name: TM111 Recombinant Rabbit Monoclonal Antibody (3H4L5)
Antibody type: Monoclonal
Antigen: Other
Description: This antibody is predicted to react with Monkey, Horse, Cat, Mouse
Antibody clone number: 3H4L5
Concentration: 0.5 mg/mL

EMC3 protein structure - 702736 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of EMC3 was achieved by transfecting MCF-7 cells with EMC3 specific siRNA (Silencer® select Product # s31615 + s31613). Western blot analysis (Fig a) was performed using Membrane enriched extracts from EMC3 knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blots were probed with Anti-EMC3 Recombinant Rabbit Monoclonal Antibody (Product # 702736, 1-3 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this Western blot is shown in histogram (Fig b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to EMC3.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on Membrane enriched extracts (30 µg lysate) of MCF-7 (Lane 1), A-431 (Lane 2), NIH/3T3 (Lane 3), PC-3 (Lane 4), HEK-293 (Lane 5), U-2 OS (Lane 6), Caco-2 (Lane 7) and A549 (Lane 8). The blots were probed with Anti-EMC3 Recombinant Rabbit Monoclonal Antibody (Product # 702736, 2.5 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 30 kDa band corresponding to EMC3 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0322BOX), 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® 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: Western blot analysis was performed on Membrane enriched extracts (30 µg lysate) of MCF-7 (Lane 1), A-431 (Lane 2), NIH/3T3 (Lane 3), PC-3 (Lane 4), HEK-293 (Lane 5), U-2 OS (Lane 6), Caco-2 (Lane 7) and A549 (Lane 8). The blots were probed with Anti-EMC3 Recombinant Rabbit Monoclonal Antibody (Product # 702736, 2.5 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 30 kDa band corresponding to EMC3 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0322BOX), 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® 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: For immunofluorescence analysis, HepG2 cells were fixed for detection of endogenous EMC3 using Anti- EMC3 Recombinant Rabbit Monoclonal Antibody (Product # 702736, 5 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of EMC3 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating membrane localization of EMC3. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Emc3 in NIH3T3 ectopically expressing Myc-tagged EMC3 cells. The cells were fixed with 4% PFA for 10 min, permeabilized with 0.2% Triton X-100 for 15 min, and blocked with blocking buffer (5% normal donkey serum in 0.02% Triton X-100/PBS) for 1hr at room temperature. Cells were stained with a Emc3 monoclonal antibody (Product # 702736, red) at a dilution of 1:100 in blocking buffer for 1.5 hours at room temperature. Ectopic expression of myc-tagged Emc3 was also detected by a c-myc antibody (green). a-tubulin and DAPI stainings were shown in purple and blue respectively. Data courtesy of Dr. Jeff Whitsett's Lab at Cincinnati Children's Hospital Medical Center.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Emc3 in NIH3T3 cells. The cells were fixed with 4% PFA for 10 min, permeabilized with 0.2% Triton X-100 for 15 min, and blocked with blocking buffer (5% normal donkey serum in 0.02% Triton X-100/PBS) for 1hr at room temperature. Cells were stained with a Emc3 monoclonal antibody (Product # 702736, red) at a dilution of 1:100 in blocking buffer for 1.5 hours at room temperature. a-tubulin and DAPI stainings were shown in purple and blue respectively. Data courtesy of Dr. Jeff Whitsett's Lab at Cincinnati Children's Hospital Medical Center.