Anti-SDHB antibody

• IHC-P

Ap

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SDHB antibody (AB151684)

IHC image of SDHB staining in normal human heart muscle formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab151684, 5μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• WB

Lab

Western blot - Anti-SDHB antibody (AB151684)

The band observed at 29 kDa could potentially be a cleaved form of SDHB due to the presence of a 28 amino acid transit peptide.

This blot was produced using a 4-12% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Bovine Serum Albumin before being incubated with ab151684 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.

All lanes:

Western blot - Anti-SDHB antibody (ab151684) at 1 µg/mL

Lane 1:

Human heart tissue lysate - total protein (<a href='/en-us/products/unavailable/human-heart-tissue-lysate-total-protein-ab29431'>ab29431</a>) at 10 µg

Lane 2:

Human Heart Mitochondrial Lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/10000 dilution

Predicted band size: 32 kDa

Observed band size: 29 kDa

true

Exposure time: 1min

• WB

Lab

Western blot - Anti-SDHB antibody (AB151684)

Lanes 1 - 3 : Merged signal (red and green). Green - ab151684 observed at 32 kDa. Red - loading control, ab130007, observed at 130 kDa.

ab151684 was shown to recognize SDHB in wild-type HEK-293 cells as signal was lost at the expected MW in SDHB knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and SDHB knockout samples were subjected to SDS-PAGE. ab151684 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1 ug/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-SDHB antibody (ab151684) at 1 µg/mL

Lane 1:

Wild-type HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

SDHB knockout HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

Lane 2:

Western blot - Human SDHB knockout HEK-293 cell line (<a href='/en-us/products/cell-lines/human-sdhb-knockout-hek-293-cell-line-ab260860'>ab260860</a>)

Lane 3:

HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

Predicted band size: 32 kDa

Observed band size: 32 kDa

false