A21972

antibody from Invitrogen Antibodies

Targeting: ECH1 HPXEL

ELISA

Immunoprecipitation

Antibody data

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Validation data

ECH1 protein structure - A21972 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of ECH1 was performed using 70% confluent log phase Hep G2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with ECH1 (9D11AF3) Mouse Monoclonal Antibody (Product # A21972) 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™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. 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: Knockdown of ECH1 was achieved by transfecting HepG2 cells with ECH1 specific siRNA (Silencer® select Cat # s4437). Immunofluorescence analysis was performed on HepG2 cells (untransfected, panel a,d), transfected with non-specific scrambled siRNA (panels b,e) and transfected with ECH1 specific siRNA (panel c,f) Cells were fixed, permeabilized, and labelled with ECH1 Mouse monoclonal Antibody (Product # A21972, 5 µg/mL), followed by Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175, 1:2000). Nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938), and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Loss of signal was observed upon siRNA mediated knockdown (panel c,f) confirming specificity of the antibody to ECH1(green). The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Human HDFn cells were paraformaldehyde fixed (4%, 20 min) and Triton X-100 permeabilized (0.1%, 15 min). The cells were incubated with the antibody at 4 µg/mL overnight at 4°C. Secondary antibody: (green) Alexa Fluor® 488 goat anti-mouse IgG (H+L) at a 1/1000 dilution for 1h. 10% Goat serum was used as the blocking agent for all blocking steps. DAPI was used to stain the cell nuclei (blue). Target protein locates mainly in mitochondria.