All lanes : Anti-PDHA1 (phospho S293) antibody (ab92696) at 1 µg/ml

All lanes : HeLa Whole Cell Lysate + Calyculin A (30 nM for 20 min)

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : goat anti-rabbit (green) and goat anti-mouse (red) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 43 kDa

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 and then treated with either buffer (lane 1) or alkaline phosphatase (lane 2), before being incubated with ab92696 overnight at 4°C. Antibody binding was detected using IR-labelled goat anti-rabbit (green) and goat anti-mouse (Red) at 1:10,000 dilution for one hour at room temperature before imaging.

 

ab92696 stained in HepG2 cells. Cells were fixed with 100% paraformaldehyde (5 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab92696 at 5 µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150120 (pseudo-colored red) and ab150081 (colored green) used at 1 ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43 µM for 1hour at room temperature

IHC image of PDHA1 (phospho S293) staining in Human lung adenocarcinoma 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 ab92696, 1µg/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.

Anti-PDHA1 (phospho S293) antibody (ab92696) at 1/1000 dilution + Human vascular smooth muscle cell whole cell lysate at 25 µg

Secondary
Polyclonal goat anti-rabbit IRDye® 800CW at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 43 kDa
Observed band size: 43 kDa


Exposure time: 5 minutes

See Abreview

Serially diluted ab92696 was bound to immobilised Phospho peptide (S293) - or Control peptide (1 microgram x mL-1). The antibody was detected by HRP-labelled goat anti-rabbit IgG

ab92696 staining PDHA1 (phospho S293) in Human HUVEC cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton X-100 pH 7.4 for 5 minutes and blocked with 5% BSA for 20 minutes at room temperature. Samples were incubated with primary antibody (1/500 in PBS) for 1 hour . A CF488-conjugated Donkey anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.

See Abreview

All lanes : Anti-PDHA1 (phospho S293) antibody (ab92696) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate
Lane 3 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate
Lane 4 : Human liver tissue lysate - total protein (ab29889)
Lane 5 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with Modified blocking peptide at 1 µg/ml
Lane 6 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate with Modified blocking peptide at 1 µg/ml
Lane 7 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate with Modified blocking peptide at 1 µg/ml
Lane 8 : Human liver tissue lysate - total protein (ab29889) with Modified blocking peptide at 1 µg/ml
Lane 9 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate with Non-modified blocking peptide at 1 µg/ml
Lane 10 : HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate with Non-modified blocking peptide at 1 µg/ml
Lane 11 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate with Non-modified blocking peptide at 1 µg/ml
Lane 12 : Human liver tissue lysate - total protein (ab29889) with Non-modified blocking peptide at 1 µg/ml

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 43 kDa
Observed band size: 43 kDa
Additional bands at: 34 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 2 minutes