Anti-IDH1 antibody [EPR21002]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IDH1 antibody [EPR21002] (AB230949)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IDH1 antibody [EPR21002] (AB230949)

Immunohistochemical analysis of paraffin-embedded human glioblastoma tissue labeling IDH1 with ab230949 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Counter stained with hematoxylin. Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP polymer) (ab214880) Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. Positive staining on human glioblastoma. The section was incubated with ab230949 at 4°C overnight.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-IDH1 antibody [EPR21002] (AB230949)

Intracellular 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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IDH1 antibody [EPR21002] (AB230949)

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.

• IP

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Immunoprecipitation - Anti-IDH1 antibody [EPR21002] (AB230949)

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.

All lanes:

Immunoprecipitation - Anti-IDH1 antibody [EPR21002] (ab230949)

Predicted band size: 46 kDa

false

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IDH1 antibody [EPR21002] (AB230949)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-IDH1 antibody [EPR21002] (AB230949)

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.

• WB

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Western blot - Anti-IDH1 antibody [EPR21002] (AB230949)

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

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - 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:

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 1/100000 dilution

Predicted band size: 46 kDa

Observed band size: 47 kDa

false

• WB

Supplier Data

Western blot - Anti-IDH1 antibody [EPR21002] (AB230949)

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:

Western blot - Anti-IDH1 antibody [EPR21002] (ab230949) at 1/1000 dilution

Lane 1:

Wild type HAP1 whole cell lysate at 10 µg

Lane 2:

IDH1 knockout HAP1 whole cell lysate at 10 µg

Lane 3:

SH-SY5Y (human neuroblastoma epithelial cell), whole cell lysate at 10 µg

Predicted band size: 46 kDa

false

Exposure time: 62s

• WB

Lab

Western blot - Anti-IDH1 antibody [EPR21002] (AB230949)

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.

All lanes:

Western blot - Anti-IDH1 antibody [EPR21002] (ab230949) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human IDH1 knockout HeLa cell lysate (<a href='/en-us/products/cell-lysates/human-idh1-knockout-hela-cell-lysate-ab257221'>ab257221</a>) at 20 µg

Secondary

Lanes 1 - 2:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Lanes 1 - 2:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Predicted band size: 46 kDa

Observed band size: 46 kDa,37 kDa

false