GAPDH Recombinant Rabbit Monoclonal Antibody [SA30-01] - Loading control

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Catalog# ET1601-4

GAPDH Recombinant Rabbit Monoclonal Antibody [SA30-01] - Loading control

Application

WB
IF-Cell
IF-Tissue
IHC-P
FC
IP

Reactivity

Human
Mouse
Rat
Chicken
Drosophila melanogaster

Predicted reactivity

Zebrafish

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

Overview

Product Name

GAPDH Recombinant Rabbit Monoclonal Antibody [SA30-01] - Loading control

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

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

Species Reactivity

Human, Mouse, Rat, Chicken, Drosophila melanogaster (Predicted: Zebrafish)

Validated Applications

WB, IF-Cell, IF-Tissue, IHC-P, FC, IP

Molecular Weight

Predicted band size: 36 kDa

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, SH-SY5Y cell lysate, NIH/3T3 cell lysate, SK-Br-3 cell lysate, rat brain tissue lysate, A549, HepG2, human liver tissue, mouse liver tissue, mouse spleen tissue, DF-1 cell lysate.

Conjugation

unconjugated

Clone Number

SA30-01

RRID

AB_3069615

Product Features

Form

Liquid

Concentration

1ug/ul

Storage Instructions

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

Storage Buffer

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

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

WB
1:50,000-1:200,000

IF-Cell
1:1,000

IF-Tissue
1:200-1:500

IHC-P
1:1,000

FC
1:1,000

IP
1-2μg/sample

Applications in Publications

WB	See 300 publications below
COIP	See 1 publications below
IF	See 1 publications below

Species in Publications

Human	See 133 publications below
Mouse	See 102 publications below
Rat	See 35 publications below

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 (such as ceruplasmin) and suppresses their translation.

Background References

1. "High-resolution structure of human D-glyceraldehyde-3-phosphate dehydrogenase."Jenkins J.L., Tanner J.J.Acta Crystallogr. D 62:290-301(2006)

2. "Structural analysis of human liver glyceraldehyde-3-phosphate dehydrogenase."Ismail S.A., Park H.W.Acta Crystallogr. D 61:1508-1513(2005)

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, cytosol, Nucleus perinuclear region, Membrane, cytoskeleton.

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 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.
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, 1/100,000) 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.

Positive control:
Lane 1: Rat liver tissue lysate, 20 µg/Lane
Lane 2: Rat lung tissue lysate, 20 µg/Lane
Lane 3: Rat lung tissue lysate, 20 µg/Lane
Lane 4: Rat heart tissue lysate, 20 µg/Lane
Lane 5: Rat cerebellum tissue lysate, 20 µg/Lane
Lane 6: Rat skeletal muscle tissue lysate, 20 µg/Lane
Lane 7: Rat spleen tissue lysate, 20 µg/Lane
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.

Cell lysates at 10 µg/Lane.

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

Exposure time: 1 minute;

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 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.
Western blot analysis of GAPDH on DF-1 cell lysates with Rabbit anti-GAPDH antibody (ET1601-4) at 1/100,000 dilution.

Cell lysates at 15 µg/Lane.

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

Exposure time: 1 second;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 2 hour at room temperature. The primary antibody (ET1601-4) at 1/100,000 dilution was used in 5% NFDM/TBST at 4 ℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1/50,000 dilution was used for 2 hour at room temperature.
Western blot analysis of GAPDH on zebrafish tissue lysates with Rabbit anti-GAPDH antibody (ET1601-4) at 1/50,000 dilution.

Lysates/proteins at 40 µg/Lane.

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

Exposure time: 14 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 (ET1601-4) at 1/50,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
Western blot analysis of GAPDH on fruit flies tissue lysates with Rabbit anti-GAPDH antibody (ET1601-4) at 1/100,000 dilution.

Lysates/proteins at 8 µg/Lane.

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

Exposure time: 20 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 (ET1601-4) at 1/100,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
Immunohistochemical analysis of paraffin-embedded human liver tissue with Rabbit anti-GAPDH antibody (ET1601-4) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) 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 (ET1601-4) 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 Rabbit anti-GAPDH antibody (ET1601-4) at 1/5,000 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 (ET1601-4) at 1/5,000 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 Rabbit anti-GAPDH antibody (ET1601-4) at 1/5,000 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 (ET1601-4) at 1/5,000 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.
Immunocytochemistry analysis of HeLa cells labeling GAPDH with Rabbit anti-GAPDH antibody (ET1601-4) at 1/1,000 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 Rabbit anti-GAPDH antibody (ET1601-4) at 1/1,000 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) 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 (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
Immunocytochemistry analysis of NIH/3T3 cells labeling GAPDH with Rabbit anti-GAPDH antibody (ET1601-4) at 1/2,500 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 Rabbit anti-GAPDH antibody (ET1601-4) at 1/2,500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) 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 (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
Flow cytometric analysis of HeLa cells labeling GAPDH.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1601-4, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
GAPDH was immunoprecipitated from 0.2 mg A549 cell lysate with ET1601-4 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using ET1601-4 at 1/20,000 dilution. HRP Conjugated Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: A549 cell lysate (input)
Lane 2: ET1601-4 IP in A549 cell lysate
Lane 3: Rabbit IgG instead of ET1601-4 in A549 cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 7 seconds; ECL: K1801