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GAPDH Mouse Monoclonal Antibody [5-E10]

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Catalog# EM1101

GAPDH Mouse Monoclonal Antibody [5-E10]

Application

  • WB

  • IF-Cell

  • IHC-P

Reactivity

  • Human

  • Mouse

  • Rat

  • Zebrafish

  • Rabbit

Conjugation

  • unconjugated

This product has been cited in peer reviewed publications, see list HERE

Overview

Product Name

GAPDH Mouse Monoclonal Antibody [5-E10]

Antibody Type

Mouse Monoclonal Antibody

Immunogen

Synthetic peptide within human GAPDH aa 10-50.

Species Reactivity

Human, Mouse, Rat, Zebrafish, Rabbit

Validated Applications

WB, IF-Cell, IHC-P

Molecular Weight

Predicted band size: 36 kDa

Positive Control

MDA-MB-231 cell lysate, HeLa cell lysate, MCF7 cell lysate, SK-Br-3 cell lysate, JAR cell lysate, NIH/3T3 cell lysate, C2C12 cell lysate, PC-12 cell lysate, Mouse kidney tissue lysate, Rat liver tissue lysate, HeLa, F9, zebrafish cell/tissue lysates, human liver tissue, mouse liver tissue, human spleen tissue, rat liver tissue, rat spleen tissue.

Conjugation

unconjugated

Clone Number

5-E10

RRID

AB_2811078

Product Features

Form

Liquid

Concentration

Storage Instructions

Shipped at 4℃. Store at +4℃ short term (1-2 weeks). It is recommended to aliquot into single-use upon delivery. Store at -20℃ long term.

Storage Buffer

1*PBS (pH7.4), 0.2% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Isotype

IgM

Purification Method

Protein L affinity purified.

Application Dilution

  • WB

  • 1:10,000-1:50,000

  • IF-Cell

  • 1:200-1:500

  • IHC-P

  • 1:200-1:1,000

Target

Function

GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) has both glyceraldehyde-3-phosphate dehydrogenase and nitrosylase activities, thereby playing a role in glycolysis and nuclear functions, respectively. It participates in nuclear events including transcription, RNA transport, DNA replication and apoptosis. GAPDH is a key enzyme in glycolysis that catalyzes the first step of the pathway by converting D-glyceraldehyde 3-phosphate (G3P) into 3-phospho-D-glyceroyl phosphate.

Background References

1. "The glyceraldehyde 3 phosphate dehydrogenase gene family: structure of a human cDNA and of an X chromosome linked pseudogene; amazing complexity of the gene family in mouse." Hanauer A., Mandel J.-L. EMBO J. 3:2627-2633(1983)

2. "Enhanced expression of a glyceraldehyde-3-phosphate dehydrogenase gene in human lung cancers." Tokunaga K., Nakamura Y., Sakata K., Fujimori K., Ohkubo M., Sawada K., Sakiyama S. Cancer Res. 47:5616-5619(1986)

3. "Glyceraldehyde-3-phosphate dehydrogenase is phosphorylated by protein kinase Ciota /lambda and plays a role in microtubule dynamics in the early secretory pathway." Tisdale E.J.J. Biol. Chem. 277:3334-3341(2001)

Sequence Similarity

Belongs to the glyceraldehyde-3-phosphate dehydrogenase family.

Post-translational Modification

S-nitrosylation of Cys-152 leads to interaction with SIAH1, followed by translocation to the nucleus (By similarity). S-nitrosylation of Cys-247 is induced by interferon-gamma and LDL(ox) implicating the iNOS-S100A8/9 transnitrosylase complex and seems to prevent interaction with phosphorylated RPL13A and to interfere with GAIT complex activity.; ISGylated.; Sulfhydration at Cys-152 increases catalytic activity.; Oxidative stress can promote the formation of high molecular weight disulfide-linked GAPDH aggregates, through a process called nucleocytoplasmic coagulation. Such aggregates can be observed in vivo in the affected tissues of patients with Alzheimer disease or alcoholic liver cirrhosis, or in cell cultures during necrosis. Oxidation at Met-46 may play a pivotal role in the formation of these insoluble structures. This modification has been detected in vitro following treatment with free radical donor (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide. It has been proposed to destabilize nearby residues, increasing the likelihood of secondary oxidative damages, including oxidation of Tyr-45 and Met-105. This cascade of oxidations may augment GAPDH misfolding, leading to intermolecular disulfide cross-linking and aggregation.; Succination of Cys-152 and Cys-247 by the Krebs cycle intermediate fumarate, which leads to S-(2-succinyl)cysteine residues, inhibits glyceraldehyde-3-phosphate dehydrogenase activity. Fumarate concentration as well as succination of cysteine residues in GAPDH is significantly increased in muscle of diabetic mammals. It was proposed that the S-(2-succinyl)cysteine chemical modification may be a useful biomarker of mitochondrial and oxidative stress in diabetes and that succination of GAPDH and other thiol proteins by fumarate may contribute to the metabolic changes underlying the development of diabetes complications.

Subcellular Location

Cytoplasm

UNIPROT

SwissProt: P04406 Human

SwissProt: P16858 Mouse

SwissProt: P04797 Rat

Synonyms

38 kDa BFA-dependent ADP-ribosylation substrate antibody

aging associated gene 9 protein antibody

Aging-associated gene 9 protein antibody

BARS-38 antibody

cb609 antibody

EC 1.2.1.12 antibody

Epididymis secretory sperm binding protein Li 162eP antibody

G3P_HUMAN antibody

G3PD antibody

G3PDH antibody

Expand

Images

  • Western blot analysis of GAPDH on different lysates with Mouse anti-GAPDH antibody (<a href="/products/EM1101" style="font-weight: bold;text-decoration: underline;">EM1101</a>) at 1/10,000 dilution.<br /><br />Lane 1: MDA-MB-231 cell lysate (20 µg/Lane)<br />Lane 2: HeLa cell lysate (20 µg/Lane)<br />Lane 3: MCF7 cell lysate (20 µg/Lane)<br />Lane 4: SK-Br-3 cell lysate (20 µg/Lane)<br />Lane 5: JAR cell lysate (20 µg/Lane)<br />Lane 6: NIH/3T3 cell lysate (20 µg/Lane)<br />Lane 7: C2C12 cell lysate (20 µg/Lane)<br />Lane 8: PC-12 cell lysate (20 µg/Lane)<br />Lane 9: Mouse kidney tissue lysate (40 µg/Lane)<br />Lane 10: Rat liver tissue lysate (40 µg/Lane)<br /><br />Predicted band size: 36 kDa<br />Observed band size: 36 kDa<br /><br />Exposure time: 1 minute 30 seconds;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/EM1101" style="font-weight: bold;text-decoration: underline;">EM1101</a>) at 1/10,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (<a href="/products/HA1006" style="font-weight: bold;text-decoration: underline;">HA1006</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of GAPDH on different lysates with Mouse anti-GAPDH antibody (EM1101) at 1/10,000 dilution.

    Lane 1: MDA-MB-231 cell lysate (20 µg/Lane)
    Lane 2: HeLa cell lysate (20 µg/Lane)
    Lane 3: MCF7 cell lysate (20 µg/Lane)
    Lane 4: SK-Br-3 cell lysate (20 µg/Lane)
    Lane 5: JAR cell lysate (20 µg/Lane)
    Lane 6: NIH/3T3 cell lysate (20 µg/Lane)
    Lane 7: C2C12 cell lysate (20 µg/Lane)
    Lane 8: PC-12 cell lysate (20 µg/Lane)
    Lane 9: Mouse kidney tissue lysate (40 µg/Lane)
    Lane 10: Rat liver tissue lysate (40 µg/Lane)

    Predicted band size: 36 kDa
    Observed band size: 36 kDa

    Exposure time: 1 minute 30 seconds;

    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (EM1101) at 1/10,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/50,000 dilution was used for 1 hour at room temperature.

  • Immunocytochemistry analysis of HeLa cells labeling GAPDH with Mouse anti-GAPDH antibody (<a href="/products/EM1101" style="font-weight: bold;text-decoration: underline;">EM1101</a>) at 1/100 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Mouse anti-GAPDH antibody (<a href="/products/EM1101" style="font-weight: bold;text-decoration: underline;">EM1101</a>) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 488, <a href="/products/HA1125" style="font-weight: bold;text-decoration: underline;">HA1125</a>) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.<br /><br />beta Tubulin (<a href="/products/ET1602-4" style="font-weight: bold;text-decoration: underline;">ET1602-4</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 594, <a href="/products/HA1122" style="font-weight: bold;text-decoration: underline;">HA1122</a>) were used as the secondary antibody at 1/1,000 dilution.

    Immunocytochemistry analysis of HeLa cells labeling GAPDH with Mouse anti-GAPDH antibody (EM1101) at 1/100 dilution.

    Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Mouse anti-GAPDH antibody (EM1101) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor™ 488, HA1125) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

    beta Tubulin (ET1602-4, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor™ 594, HA1122) were used as the secondary antibody at 1/1,000 dilution.

  • Immunocytochemistry analysis of F9 cells labeling GAPDH with Mouse anti-GAPDH antibody (<a href="/products/EM1101" style="font-weight: bold;text-decoration: underline;">EM1101</a>) at 1/100 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Mouse anti-GAPDH antibody (<a href="/products/EM1101" style="font-weight: bold;text-decoration: underline;">EM1101</a>) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 488, <a href="/products/HA1125" style="font-weight: bold;text-decoration: underline;">HA1125</a>) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.<br /><br />beta Tubulin (<a href="/products/ET1602-4" style="font-weight: bold;text-decoration: underline;">ET1602-4</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 594, <a href="/products/HA1122" style="font-weight: bold;text-decoration: underline;">HA1122</a>) were used as the secondary antibody at 1/1,000 dilution.

    Immunocytochemistry analysis of F9 cells labeling GAPDH with Mouse anti-GAPDH antibody (EM1101) at 1/100 dilution.

    Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Mouse anti-GAPDH antibody (EM1101) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Mouse IgG H&L (iFluor™ 488, HA1125) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

    beta Tubulin (ET1602-4, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Rabbit IgG H&L (iFluor™ 594, HA1122) were used as the secondary antibody at 1/1,000 dilution.

  • Western blot analysis of GAPDH on zebrafish cell/tissue lysates with Mouse anti-GAPDH antibody (<a href="/products/EM1101" style="font-weight: bold;text-decoration: underline;">EM1101</a>) at 1/10,000 dilution.<br /><br />Lysates/proteins at 20 µg/Lane.<br /><br />Predicted band size: 36 kDa<br />Observed band size: 36 kDa<br /><br />Exposure time: 14 seconds; ECL: K1801;<br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/EM1101" style="font-weight: bold;text-decoration: underline;">EM1101</a>) at 1/10,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Mouse IgG - HRP Secondary Antibody (<a href="/products/HA1006" style="font-weight: bold;text-decoration: underline;">HA1006</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of GAPDH on zebrafish cell/tissue lysates with Mouse anti-GAPDH antibody (EM1101) at 1/10,000 dilution.

    Lysates/proteins at 20 µg/Lane.

    Predicted band size: 36 kDa
    Observed band size: 36 kDa

    Exposure time: 14 seconds; ECL: K1801;
    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (EM1101) at 1/10,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1/50,000 dilution was used for 1 hour at room temperature.

  • Immunohistochemical analysis of paraffin-embedded human liver tissue with Mouse anti-GAPDH antibody (<a href="/products/EM1101" style="font-weight: bold;text-decoration: underline;">EM1101</a>) at 1/200 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/EM1101" style="font-weight: bold;text-decoration: underline;">EM1101</a>) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded human liver tissue with Mouse anti-GAPDH antibody (EM1101) at 1/200 dilution.

    The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (EM1101) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded mouse liver tissue with Mouse anti-GAPDH antibody (<a href="/products/EM1101" style="font-weight: bold;text-decoration: underline;">EM1101</a>) at 1/200 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/EM1101" style="font-weight: bold;text-decoration: underline;">EM1101</a>) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded mouse liver tissue with Mouse anti-GAPDH antibody (EM1101) at 1/200 dilution.

    The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (EM1101) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded human spleen tissue with Mouse anti-GAPDH antibody (<a href="/products/EM1101" style="font-weight: bold;text-decoration: underline;">EM1101</a>) at 1/200 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/EM1101" style="font-weight: bold;text-decoration: underline;">EM1101</a>) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded human spleen tissue with Mouse anti-GAPDH antibody (EM1101) at 1/200 dilution.

    The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (EM1101) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded rat liver tissue with Mouse anti-GAPDH antibody (<a href="/products/EM1101" style="font-weight: bold;text-decoration: underline;">EM1101</a>) at 1/200 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/EM1101" style="font-weight: bold;text-decoration: underline;">EM1101</a>) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded rat liver tissue with Mouse anti-GAPDH antibody (EM1101) at 1/200 dilution.

    The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (EM1101) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded rat spleen tissue with Mouse anti-GAPDH antibody (<a href="/products/EM1101" style="font-weight: bold;text-decoration: underline;">EM1101</a>) at 1/200 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/EM1101" style="font-weight: bold;text-decoration: underline;">EM1101</a>) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded rat spleen tissue with Mouse anti-GAPDH antibody (EM1101) at 1/200 dilution.

    The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (EM1101) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Citation

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