PRODUCT CODE: ET1601-4

GAPDH Recombinant Rabbit Monoclonal Antibody [SA30-01] (ET1601-4)

  • Zebrafish
  • Recombinant

Applications

  • WB

  • ICC/IF

  • IHC-P

  • IP

  • FC

REACTIVITY

  • Human

  • Mouse

  • Rat

  • Zebrafish

Western blot analysis of GAPDH on Hela cell lysates with Rabbit anti-GAPDH antibody (ET1601-4).<br />
<br />
Hela cell lysates at 10 µg/Lane.<br />
<br />
Predicted band size: 36 kDa<br />
Observed band size: 36 kDa<br />
<br />
12% 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 (ET1601-4) at serial dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:300,000 dilution was used for 1 hour at room temperature.
  • Western blot analysis of GAPDH on Hela cell lysates with Rabbit anti-GAPDH antibody (ET1601-4).<br />
<br />
Hela cell lysates at 10 µg/Lane.<br />
<br />
Predicted band size: 36 kDa<br />
Observed band size: 36 kDa<br />
<br />
12% 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 (ET1601-4) at serial dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:300,000 dilution was used for 1 hour at room temperature.
  • Western blot analysis of GAPDH on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1601-4, 1/5,000) was used in 5% NFDM/TBST at room temperature for 1 hour. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:200,000 dilution was used for 45 mins at room temperature.<br />
<br />
Positive control: <br />
Lane 1: PC-3 cell lysate, 10 µg/Lane<br />
Lane 2: Mouse colon tissue lysate, 20 µg/Lane<br />
Lane 3: SHSY-5Y cell lysate, 10 µg/Lane<br />
Lane 4: PC-3 cell lysate, 10 µg/Lane<br />
Lane 5: NIH/3T3 cell lysate, 10 µg/Lane<br />
Lane 6: SKBR3 cell lysate, 10 µg/Lane<br />
Lane 7: Rat brain tissue lysate, 20 µg/Lane
  • Western blot analysis of GAPDH on different lysates. Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1601-4, 1/10,000) was used in 5% NFDM/TBST at room temperature for 1 hour. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:200,000 dilution was used for 45 mins at room temperature.<br />
<br />
Positive control: <br />
Lane 1: Rat liver tissue lysate, 20 µg/Lane<br />
Lane 2: Rat lung tissue lysate, 20 µg/Lane<br />
Lane 3: Rat lung tissue lysate, 20 µg/Lane<br />
Lane 4: Rat heart tissue lysate, 20 µg/Lane<br />
Lane 5: Rat cerebellum tissue lysate, 20 µg/Lane<br />
Lane 6: Rat skeletal muscle tissue lysate, 20 µg/Lane<br />
Lane 7: Rat spleen tissue lysate, 20 µg/Lane<br />
Lane 8: Rat small intestine tissue lysate, 20 µg/Lane
  • Western blot analysis of GAPDH on different lysates with Rabbit anti-GAPDH antibody (ET1601-4) at 1/80,000 dilution.<br />
<br />
Cell lysates at 10 µg/Lane, tissue lysates at 20 µg/Lane.<br />
<br />
Predicted band size: 36 kDa<br />
Observed band size: 36 kDa<br />
<br />
Exposure time: 1 minute;<br />
<br />
12% 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 (ET1601-4) at 1/80,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:300,000 dilution was used for 1 hour at room temperature.
  • ICC staining of GAPDH in A549 cells (green). 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 (ET1601-4, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
  • ICC staining of GAPDH in HepG2 cells (green). 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 (ET1601-4, 1/50) for 1 hour at room temperature, washed with PBS. Alexa Fluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1,000 dilution. The nuclear counter stain is DAPI (blue).
  • Immunohistochemical analysis of paraffin-embedded human liver tissue using anti-GAPDH antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-4, 1/50) for 30 minutes 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 spleen tissue using anti-GAPDH antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1601-4, 1/50) for 30 minutes 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.
  • Flow cytometric analysis of GAPDH was done on HepG2 cells. The cells were fixed, permeabilized and stained with the primary antibody (ET1601-4, 1/50) (blue). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Rabbit IgG Secondary antibody at 1/1000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; red).
Western blot analysis of GAPDH on Hela cell lysates with Rabbit anti-GAPDH antibody (ET1601-4).

Hela cell lysates at 10 µg/Lane.

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

12% 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 (ET1601-4) at serial dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:300,000 dilution was used for 1 hour at room temperature.

Applications

  • WB

  • ICC/IF

  • IHC-P

  • IP

  • FC

REACTIVITY

  • Human

  • Mouse

  • Rat

  • Zebrafish

SPECIFICATIONS

Product Type

Recombinant Rabbit monoclonal primary

Product Name

GAPDH Recombinant Rabbit Monoclonal Antibody [SA30-01] (ET1601-4)

Immunogen

Recombinant protein within mouse gapdh aa 94-333 / 333.

Host

Rabbit

Positive Control

Rat liver tissue lysate, rat lung tissue lysate, rat heart tissue lysate, rat cerebellum tissue lysate, rat skeletal muscle tissue lysate, rat spleen tissue lysate, rat small intestine tissue lysate, hybrid fish (crucian-carp) brain tissue lysates, PC-3 cell lysate, mouse colon tissue lysate, SHSY-5Y cell lysate, NIH/3T3 cell lysate, SKBR3 cell lysate, rat brain tissue lysate, A549, HepG2, human liver tissue, mouse liver tissue, mouse spleen tissue.

Conjugation

Unconjugated

Clonality

Monoclonal

Clone Number

SA30-01

PROPERTIES

Form

Liquid

Storage Condition

Store at +4C after thawing. Aliquot store at -20C or -80C. Avoid repeated freeze / thaw cycles.

Storage Buffer

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

Concentration

1ug/ul

PURIFICATION

Protein A affinity purified.

MOLECULAR WEIGHT

36 kDa

Isotype

IgG

APPLICATION DILUTION

  • WB

  • 1:5,000-1:640,000

  • ICC/IF

  • 1:50-1:200

  • IHC-P

  • 1:50-1:200

  • FC

  • 1:50-1:100

  • IP

  • Use at an assay dependent concentration.

TARGET

UNIPROT #

PROTEIN NAME

GAPDH

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;GAPD antibody;GAPDH antibody;Glyceraldehyde 3 phosphate dehydrogenase antibody;Glyceraldehyde-3-phosphate dehydrogenase antibody;HEL-S-162eP antibody;KNC-NDS6 antibody;MGC102544 antibody;MGC102546 antibody;MGC103190 antibody;MGC103191 antibody;MGC105239 antibody;MGC127711 antibody;MGC88685 antibody;OCAS, p38 component antibody;OCT1 coactivator in S phase, 38-KD component antibody;peptidyl cysteine S nitrosylase GAPDH antibody;Peptidyl-cysteine S-nitrosylase GAPDH antibody;wu:fb33a10 antibody

SEQUENCE SIMILARITIES

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

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 (such as ceruplasmin) and suppresses their translation.

CITATIONS

  • Bin Yu;Xianrong Zhang

    Antenatal Corticosteroid Therapy Attenuates Angiogenesis Through Inhibiting Osteoclastogenesis in Young Mice

  • Zhang, D., Zhang, J. T., Pa...

    Syndecan-1 Shedding by Matrix Metalloproteinase-9 Signaling Regulates Alveolar Epithelial Tight Junction in Lipopolysaccharide-Induced Early Acute Lung Injury

  • Jie Wu ;Ayaz Najafov;Honggu...

    Pharmacological targeting of MCL-1 promotes mitophagy and improves disease pathologies in an Alzheimer’s disease mouse model

  • Wu, Y., Xue, J., Li, Y., Wu...

    Expression, clinical significance and correlation of RUNX3 and HER2 in colorectal cancer

  • He, J., Yang, J., Chen, L.,...

    SGK1‐targeted TRPV1 regulates bladder smooth muscle cell proliferation due to BOO in mice via NFAT2

  • Liu, W., Zhang, L., Sun, S....

    Cordycepin inhibits inflammatory responses through suppression of ERK activation in zebrafish. Developmental and comparative immunology, 124, 104178. Advance online publication.

  • Li, S., Li, L., Lin, X., Ch...

    Targeted Inhibition of Tumor Inflammation and Tumor-Platelet Crosstalk by Nanoparticle-Mediated Drug Delivery Mitigates Cancer Metastasis

  • Ye, Dan et al.

    MicroRNA-125a-mediated regulation of the mevalonate signaling pathway contributes to high glucose-induced proliferation and migration of vascular smooth muscle cells. | Molecular Medicine Reports [2020]

  • Yin, P., Li, D., Zhao, Q., ...

    Gsα deficiency facilitates cardiac remodeling via CREB/ Bmp10-mediated signaling

  • Li, Ronggang et al.

    Theaflavin attenuates cerebral ischemia/reperfusion injury by abolishing miRNA-128-3p-mediated Nrf2 inhibition and reducing oxidative stress. | Molecular Medicine Reports [2019]

  • Zhang, Y., Huang, Q., Wang,...

    The Food Additive β-Caryophyllene Exerts Its Neuroprotective Effects Through the JAK2-STAT3-BACE1 Pathway

  • Zhang, F., Wang, B., Liu, S...

    Bacillus subtilis revives conventional antibiotics against Staphylococcus aureus osteomyelitis. Microbial cell factories, 20(1), 102.

  • Jian Huang

    Tumor-derived HMGB1 induces CD62Ldim neutrophil polarization and promotes lung metastasis in triple-negative breast cancer