Anti-PDHA1 (phospho S293) antibody

• WB

Lab

Western blot - Anti-PDHA1 (phospho S293) antibody (AB92696)

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.

All lanes:

Western blot - Anti-PDHA1 (phospho S293) antibody (ab92696) at 1 µg/mL

All lanes:

HeLa Whole Cell Lysate + Calyculin A (30 nM for 20 min) at 20 µg

Secondary

All lanes:

goat anti-rabbit (green) and goat anti-mouse (red) at 1/10000 dilution

Predicted band size: 43 kDa

false

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PDHA1 (phospho S293) antibody (AB92696)

ab92696 staining PDHA1 (phospho S293) in HepG2 cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab92696 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PDHA1 (phospho S293) antibody (AB92696)

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.

• ICC/IF

AbReview39449****

Immunocytochemistry/ Immunofluorescence - Anti-PDHA1 (phospho S293) antibody (AB92696)

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.

This image is courtesy of an Abreview submitted by Dimitra Kalamida

• ELISA

Lab

ELISA - Anti-PDHA1 (phospho S293) antibody (AB92696)

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

• WB

AbReview55569****

Western blot - Anti-PDHA1 (phospho S293) antibody (AB92696)

All lanes:

Western blot - Anti-PDHA1 (phospho S293) antibody (ab92696) at 1/1000 dilution

All lanes:

Human vascular smooth muscle cell whole cell lysate at 25 µg

Secondary

All lanes:

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

Predicted band size: 43 kDa

Observed band size: 43 kDa

false

Exposure time: 5min

This image is courtesy of an anonymous abreview.

• WB

Unknown

Western blot - Anti-PDHA1 (phospho S293) antibody (AB92696)

All lanes:

Western blot - Anti-PDHA1 (phospho S293) antibody (ab92696) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 2:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg

Lane 3:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 10 µg

Lane 4:

Human liver tissue lysate - total protein (<a href='/en-us/products/unavailable/human-liver-tissue-lysate-total-protein-ab29889'>ab29889</a>) at 10 µg

Lane 5:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg with Modified blocking peptide

Lane 6:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg with Modified blocking peptide

Lane 7:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 10 µg with Modified blocking peptide

Lane 8:

Human liver tissue lysate - total protein (<a href='/en-us/products/unavailable/human-liver-tissue-lysate-total-protein-ab29889'>ab29889</a>) at 10 µg with Modified blocking peptide

Lane 9:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg with Non-modified blocking peptide

Lane 10:

HepG2 (Human hepatocellular liver carcinoma cell line) Whole Cell Lysate at 10 µg with Non-modified blocking peptide

Lane 11:

HEK293 (Human embryonic kidney cell line) Whole Cell Lysate at 10 µg with Non-modified blocking peptide

Lane 12:

Human liver tissue lysate - total protein (<a href='/en-us/products/unavailable/human-liver-tissue-lysate-total-protein-ab29889'>ab29889</a>) at 10 µg with Non-modified blocking peptide

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 43 kDa

Observed band size: 34 kDa,43 kDa

true

Exposure time: 2min