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GAPDH Mouse Monoclonal Antibody [12D6] - Loading control

Usd: 350

Specification

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Catalog# M1310-2

GAPDH Mouse Monoclonal Antibody [12D6] - Loading control

Application

  • WB

  • IF-Cell

  • IHC-P

Reactivity

  • Human

  • Mouse

  • Rat

  • Zebrafish

  • Escherichia coli

Conjugation

  • unconjugated

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

Overview

Product Name

GAPDH Mouse Monoclonal Antibody [12D6] - Loading control

Antibody Type

Mouse Monoclonal Antibody

Immunogen

Synthetic peptide within human GAPDH aa 180-220.

Species Reactivity

Human, Mouse, Rat, Zebrafish, Escherichia coli

Validated Applications

WB, IF-Cell, IHC-P

Molecular Weight

Predicted band size: 36 kDa

Positive Control

Hela cell lysate, A549 cell lysate, HepG2 cell lysate, PC-12 cell lysate, F9 cell lysate, Escherichia coli lysate, D3, A431, zebrafish tissue lysates, human thyroid carcinoma tissue, human colon carcinoma tissue.

Conjugation

unconjugated

Clone Number

12D6

RRID

AB_3073066

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

IgG1

Purification Method

Protein G affinity purified.

Application Dilution

  • WB

  • 1:2000-1:5,000

  • IF-Cell

  • 1:200

  • IHC-P

  • 1:600

Target

Function

Has both glyceraldehyde-3-phosphate dehydrogenase and nitrosylase activities, thereby playing a role in glycolysis and nuclear functions, respectively. Participates in nuclear events including transcription, RNA transport, DNA replication and apoptosis. Nuclear functions are probably due to the nitrosylase activity that mediates cysteine S-nitrosylation of nuclear target proteins such as SIRT1, HDAC2 and PRKDC. Modulates the organization and assembly of the cytoskeleton. Facilitates the CHP1-dependent microtubule and membrane associations through its ability to stimulate the binding of CHP1 to microtubules. Glyceraldehyde-3-phosphate dehydrogenase 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. Component of the GAIT (gamma interferon-activated inhibitor of translation) complex which mediates interferon-gamma-induced transcript-selective translation inhibition in inflammation processes. Upon interferon-gamma treatment assembles into the GAIT complex which binds to stem loop-containing GAIT elements in the 3'-UTR of diverse inflammatory mRNAs and suppresses their translation.

Background References

1. Cheng, Y., Hou, T., Ping, J., Chen, T., & Yin, B. (2018). LMO3 promotes hepatocellular carcinoma invasion, metastasis and anoikis inhibition by directly interacting with LATS1 and suppressing Hippo signaling. J Exp Clin Cancer Res, 37(1), 228.

2. Liu, J., Wang, Y., Song, L., Zeng, L., Yi, W., Liu, T., . . . Cong, Y. S. (2017). A critical role of DDRGK1 in endoplasmic reticulum homoeostasis via regulation of IRE1alpha stability. Nat Commun, 8, 14186.

3. Jiang, Y., Tian, M., Lin, W., Wang, X., & Wang, X. (2018). Protein Kinase Serine/Threonine Kinase 24 Positively Regulates Interleukin 17-Induced Inflammation by Promoting IKK Complex Activation. Front Immunol, 9, 921.

4. Bin, G., Jiarong, Z., Shihao, W., Xiuli, S., Cheng, X., Liangbiao, C., & Ming, Z. (2012). Aire promotes the self-renewal of embryonic stem cells through Lin28. Stem Cells Dev, 21(15), 2878-2890.

5. Liu, J., Wang, Y., Song, L., et al. Cong, Y. S. (2017). A critical role of DDRGK1 in endoplasmic reticulum homoeostasis via regulation of IRE1alpha stability. Nat Commun, 8, 14186. doi: 10.1038/ncomms14186

6. Zhang, D., Zhao, Q., Sun, H., Yin, L., Wu, J., Xu, J., . . . Liang, C. (2016). Defective autophagy leads to the suppression of stem-like features of CD271(+) osteosarcoma cells. J Biomed Sci, 23(1), 82. doi: 10.1186/s12929-016-0297-5

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, Nucleus, Membrane.

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 cells lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (<a href="/products/M1310-2" style="font-weight: bold;text-decoration: underline;">M1310-2</a>, 1/1000) was used in 5% BSA 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 1:5,000 dilution was used for 1 hour at room temperature.<br /><span style="font-weight: bold;">Positive control:</span> <br />Lane 1: Hela cell lysate<br />Lane 2: A549 cell lysate<br />Lane 3: HepG2 cell lysate<br />Lane 4: PC-12 cell lysate<br />Lane 5: F9 cell lysate

    Western blot analysis of GAPDH on different cells lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (M1310-2, 1/1000) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1:5,000 dilution was used for 1 hour at room temperature.
    Positive control:
    Lane 1: Hela cell lysate
    Lane 2: A549 cell lysate
    Lane 3: HepG2 cell lysate
    Lane 4: PC-12 cell lysate
    Lane 5: F9 cell lysate

  • Western blot analysis of GAPDH on zebrafish tissue lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (<a href="/products/M1310-2" style="font-weight: bold;text-decoration: underline;">M1310-2</a>, 1/500) was used in 5% BSA 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 1:5,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of GAPDH on zebrafish tissue lysates. Proteins were transferred to a PVDF membrane and blocked with 5% BSA in PBS for 1 hour at room temperature. The primary antibody (M1310-2, 1/500) was used in 5% BSA at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1:5,000 dilution was used for 1 hour at room temperature.

  • Western blot analysis of GAPDH on different lysates with Mouse anti-GAPDH antibody (<a href="/products/M1310-2" style="font-weight: bold;text-decoration: underline;">M1310-2</a>) at 1/500 dilution.<br /><br />Lane 1: Escherichia coli lysate<br />Lane 2: Escherichia coli lysate<br /><br />Lysates/proteins at 10 µg/Lane.<br /><br />Predicted band size: 36 kDa<br />Observed band size: 36 kDa<br /><br />Exposure time: 1 minute;<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/M1310-2" style="font-weight: bold;text-decoration: underline;">M1310-2</a>) at 1/500 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 1:150,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of GAPDH on different lysates with Mouse anti-GAPDH antibody (M1310-2) at 1/500 dilution.

    Lane 1: Escherichia coli lysate
    Lane 2: Escherichia coli lysate

    Lysates/proteins at 10 µg/Lane.

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

    Exposure time: 1 minute;

    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 (M1310-2) at 1/500 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Mouse IgG - HRP Secondary Antibody (HA1006) at 1:150,000 dilution was used for 1 hour at room temperature.

  • ICC staining of GAPDH in D3 cells (red). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (<a href="/products/M1310-2" style="font-weight: bold;text-decoration: underline;">M1310-2</a>, 1/100) for 1 hour at room temperature, washed with PBS. Alexa Fluor&reg;555 Goat anti-Mouse IgG was used as the secondary antibody at 1/100 dilution. The nuclear counter stain is DAPI (blue).

    ICC staining of GAPDH in D3 cells (red). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (M1310-2, 1/100) for 1 hour at room temperature, washed with PBS. Alexa Fluor®555 Goat anti-Mouse IgG was used as the secondary antibody at 1/100 dilution. The nuclear counter stain is DAPI (blue).

  • ICC staining of GAPDH in A431 cells (red). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (<a href="/products/M1310-2" style="font-weight: bold;text-decoration: underline;">M1310-2</a>, 1/100) for 1 hour at room temperature, washed with PBS. Alexa Fluor&reg;555 Goat anti-Mouse IgG was used as the secondary antibody at 1/100 dilution. The nuclear counter stain is DAPI (blue).

    ICC staining of GAPDH in A431 cells (red). Formalin fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature and blocked with 1% Blocker BSA for 15 minutes at room temperature. Cells were probed with the primary antibody (M1310-2, 1/100) for 1 hour at room temperature, washed with PBS. Alexa Fluor®555 Goat anti-Mouse IgG was used as the secondary antibody at 1/100 dilution. The nuclear counter stain is DAPI (blue).

  • Immunohistochemical analysis of paraffin-embedded human thyroid carcinoma tissue with Mouse anti-GAPDH antibody (<a href="/products/M1310-2" style="font-weight: bold;text-decoration: underline;">M1310-2</a>) at 1/600 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 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/M1310-2" style="font-weight: bold;text-decoration: underline;">M1310-2</a>) at 1/600 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 thyroid carcinoma tissue with Mouse anti-GAPDH antibody (M1310-2) at 1/600 dilution.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 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 (M1310-2) at 1/600 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 colon carcinoma tissue with Mouse anti-GAPDH antibody (<a href="/products/M1310-2" style="font-weight: bold;text-decoration: underline;">M1310-2</a>) at 1/600 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 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/M1310-2" style="font-weight: bold;text-decoration: underline;">M1310-2</a>) at 1/600 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 colon carcinoma tissue with Mouse anti-GAPDH antibody (M1310-2) at 1/600 dilution.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 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 (M1310-2) at 1/600 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 colon tissue with Mouse anti-GAPDH antibody (<a href="/products/M1310-2" style="font-weight: bold;text-decoration: underline;">M1310-2</a>) at 1/1,000 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/M1310-2" style="font-weight: bold;text-decoration: underline;">M1310-2</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 colon tissue with Mouse anti-GAPDH antibody (M1310-2) at 1/1,000 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 (M1310-2) 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 thyroid tissue with Mouse anti-GAPDH antibody (<a href="/products/M1310-2" style="font-weight: bold;text-decoration: underline;">M1310-2</a>) at 1/1,000 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/M1310-2" style="font-weight: bold;text-decoration: underline;">M1310-2</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 thyroid tissue with Mouse anti-GAPDH antibody (M1310-2) at 1/1,000 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 (M1310-2) 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 colon tissue with Mouse anti-GAPDH antibody (<a href="/products/M1310-2" style="font-weight: bold;text-decoration: underline;">M1310-2</a>) at 1/1,000 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/M1310-2" style="font-weight: bold;text-decoration: underline;">M1310-2</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 colon tissue with Mouse anti-GAPDH antibody (M1310-2) at 1/1,000 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 (M1310-2) 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 thyroid tissue with Mouse anti-GAPDH antibody (<a href="/products/M1310-2" style="font-weight: bold;text-decoration: underline;">M1310-2</a>) at 1/1,000 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/M1310-2" style="font-weight: bold;text-decoration: underline;">M1310-2</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 thyroid tissue with Mouse anti-GAPDH antibody (M1310-2) at 1/1,000 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 (M1310-2) 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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