Anti-NDUFA9 antibody [20C11B11B11] (ab14713)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - NDUFA9 antibody [20C11] (ab14713)

Ab14713 staining human skeletal muscle. Staining is localized to the cytoplasm.
Left panel: with primary antibody at 2 ug/ml. Right panel: isotype control.
Sections were stained using an automated system (Dako PT Link), at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 antigen retrieval buffer, EDTA pH 9.0. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

  Western blot - NDUFA9 antibody [20C11] (ab14713)

All lanes : Anti-NDUFA9 antibody [20C11B11B11] (ab14713) at 1 µg/ml

Lane 1 : WI38 (Human lung fibroblast cell line) Whole Cell Lysate
Lane 2 : Testis (Human) Tissue Lysate - adult normal tissue (ab30257)
Lane 3 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate

Lysates/proteins at 10 µg per lane.

Secondary
Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 40 kDa
Observed band size : 36 kDa (why is the actual band size different from the predicted?)
Additional bands at : 58 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 20 minutes

The band observed at 36 kDa could potentially be a cleaved form of NDUFA9 due to the presence of a 35 amino acid transit peptide.

  Western blot - NDUFA9 antibody [20C11] (ab14713)

All lanes : Anti-NDUFA9 antibody [20C11B11B11] (ab14713)

Lane 1 : Isolated mitochondria from Human heart at 5 µg
Lane 2 : Isolated mitochondria from Bovine heart at 1 µg
Lane 3 : Isolated mitochondria from Rat heart at 10 µg
Lane 4 : Isolated mitochondria from Mouse Heart at 10 µg

Secondary
Goat anti-Mouse IgG

Predicted band size : 40 kDa
Observed band size : 37 kDa (why is the actual band size different from the predicted?)

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - NDUFA9 antibody [20C11] (ab14713)

ab14713 a 1/100 dilution staining NDUFA9 in Human spinal column tissue by Immunohistochemistry (Formalin/PFA-Fixed paraffin-embedded sections). Antibody was incubated with the sample for 1 hour. Sections were incubated in peroxidase-conjugated rabbit anti-mouse secondary (diluted 1/100 in 4% BSA in PBST) for 1 hour at room temperature. Sections were washed x3 in PBST and peroxidase activity was demonstrated using kit. Antigen retrieval was performed by 1 minute of pressure cooking with 1 mmol EDTA pH 8.0.