All lanes : Anti-IDH1 antibody [EPR21002] (ab230949) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : IDH1 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 46 kDa
Observed band size: 46 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab230949 observed at 46 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab230949 was shown to react with IDH1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264916 (knockout cell lysate ab257221) was used. Wild-type HeLa and IDH1 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab230949 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling IDH1 with ab230949 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic and nuclear staining in rat kidney (PMID:30153799). Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

All lanes : Anti-IDH1 antibody [EPR21002] (ab230949) at 1/1000 dilution

Lane 1 : Wild type HAP1 whole cell lysate
Lane 2 : IDH1 knockout HAP1 whole cell lysate
Lane 3 : SH-SY5Y (human neuroblastoma epithelial cell), whole cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 46 kDa


Exposure time: 62 seconds

ab230949 was shown to specifically react with IDH1 in wild-type HAP1 cells as signal was lost in IDH1 knockout cells. Wild-type and IDH1 knockout samples were subjected to SDS-PAGE. ab230949 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-IDH1 antibody [EPR21002] (ab230949) at 1/1000 dilution

Lane 1 : SH-SY5Y (human neuroblastoma cell line from bone marrow) whole cell lysate at 20 µg
Lane 2 : Raji (human Burkitt's lymphoma cell line) whole cell lysate at 20 µg
Lane 3 : C2C12 (mouse myoblast cell line) whole cell lysate at 20 µg
Lane 4 : Neuro-2a (mouse neuroblastoma cell line) whole cell lysate at 20 µg
Lane 5 : Human fetal brain lysate at 20 µg
Lane 6 : Human fetal kidney lysate at 20 µg
Lane 7 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg
Lane 8 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 9 : C6 (rat glial tumor cell line) whole cell lysate at 10 µg
Lane 10 : PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 46 kDa
Observed band size: 47 kDa why is the actual band size different from the predicted?

Exposure times: Lanes 1-6: 3 minutes; Lanes 7-8: 15 seconds; Lanes 9-10: 58 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded mouse kidney tissue labeling IDH1 with ab230949 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic and nuclear staining in mouse kidney (PMID:30153799). Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human endometrium cancer tissue labeling IDH1 with ab230949 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic and nuclear staining in human endometrium cancer (PMID:29921847). Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human stomach tissue labeling IDH1 with ab230949 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic and nucleus staining in human stomach (PMID:27466503). Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

IDH1 was immunoprecipitated from 0.35 mg of SH-SY5Y (human neuroblastoma cell line from bone marrow) whole cell lysate with ab230949 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab230949 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: SH-SY5Y whole cell lysate 10 µg (Input). 

Lane 2: ab230949 IP in SH-SY5Y whole cell lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab230949 in SH-SY5Y whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 30 seconds.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cell line labeling IDH1 with ab230949 at 1/60 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.