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  • Product Name
    GAPDH Antibody
  • Catalog No.
  • Source
  • Application
  • Reactivity
  • UniProt
  • Mol.Wt.
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Product Information

Alternative Names:Expand▼

GAPDH, A1 40 kd subunit, Activator 1 40 kd subunit, G3PD, GAPD, G3pdh, Rfc40, Rf-c 40 kd subunit


WB 1:3000-1:30000 IHC 1:200 IF 1:200, ELISA(peptide) 1:20000-1:40000








The antiserum was purified by peptide affinity chromatography using SulfoLink™ Coupling Resin (Thermo Fisher Scientific).


GAPDH antibody detects endogenous levels of total GAPDH.





Storage Condition and Buffer:

Rabbit IgG in phosphate buffered saline , pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol.Store at -20 °C.Stable for 12 months from date of receipt.

Immunogen Information


A synthesized peptide derived from human GAPDH.


>>Visit The Human Protein Atlas

Gene id:

Molecular Weight:

Observed Mol.Wt.: 37kDa.
Predicted Mol.Wt.: 37kDa.

Subcellular Location:

Cytoplasm > cytosol. Nucleus. Cytoplasm > perinuclear region. Membrane. Translocates to the nucleus following S-nitrosylation and interaction with SIAH1, which contains a nuclear localization signal (By similarity). Postnuclear and Perinuclear regions.


Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) iswell known as one of the key enzymes involved in glycolysis.As well as functioning as a glycolytic enzyme in cytoplasm,recent evidence suggests that mammalian GAPDHis also involved in a great number of intracellular procesessuch as membrane fusion, microtubule bundling, phosphotransferaseactivity, nuclear RNA export, DNA replication,and DNA repair.

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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 (By similarity). 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.

Post-translational Modifications:

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.

Subcellular Location:


Extracellular region or secreted Cytosol Plasma membrane Cytoskeleton Lysosome Endosome Peroxisome ER Golgi apparatus Nucleus Mitochondrion Manual annotation Automatic computational assertionGraphics by Christian Stolte

Subunit Structure:

Homotetramer. Interacts with TPPP; the interaction is direct. Interacts (when S-nitrosylated) with SIAH1; leading to nuclear translocation. Interacts with RILPL1/GOSPEL, leading to prevent the interaction between GAPDH and SIAH1 and prevent nuclear translocation. Interacts with CHP1; the interaction increases the binding of CHP1 with microtubules. Associates with microtubules (By similarity). Interacts with EIF1AD, USP25, PRKCI and WARS. Interacts with phosphorylated RPL13A; inhibited by oxidatively-modified low-densitity lipoprotein (LDL(ox)). Component of the GAIT complex. Interacts with FKBP6; leading to inhibit GAPDH catalytic activity.


The [IL]-x-C-x-x-[DE] motif is a proposed target motif for cysteine S-nitrosylation mediated by the iNOS-S100A8/A9 transnitrosylase complex.Belongs to the glyceraldehyde-3-phosphate dehydrogenase family.

Research Fields

Research Fields:

· Environmental Information Processing > Signal transduction > HIF-1 signaling pathway.(View pathway)
· Human Diseases > Neurodegenerative diseases > Alzheimer's disease.
· Metabolism > Global and overview maps > Biosynthesis of amino acids.
· Metabolism > Global and overview maps > Carbon metabolism.
· Metabolism > Carbohydrate metabolism > Glycolysis / Gluconeogenesis.
· Metabolism > Global and overview maps > Metabolic pathways.

Western blot analysis of extracts from rat brain, mouse muscle, rat liver, using GAPDH antibody.
Western blot analysis of extracts of various sample,using GAPDH antibody.
AF7021 at 1/50 staining human colon cancer tissue sections by IHC-P. The tissue was formaldehyde fixed and a heat mediated antigen retrieval step in citrate buffer was performed. The tissue was then blocked and incubated with the antibody for 1.5 hours at 22°C. An HRP conjugated goat anti-rabbit antibody was used as the secondary.
AF7021 at 1/50 staining human colon cancer tissue sections by IHC-P. The tissue was formaldehyde fixed and a heat mediated antigen retrieval step in citrate buffer was performed. The tissue was then blocked and incubated with the antibody for 1.5 hours at 22°C. An HRP conjugated goat anti-rabbit antibody was used as the secondary.
AF7021 staining Hela by IF/ICC. The sample were fixed with PFA and permeabilized in 0.1% Triton X-100,then blocked in 10% serum for 45 minutes at 25°C. The primary antibody was diluted at 1/200 and incubated with the sample for 1 hour at 37°C. An Alexa Fluor 594 conjugated goat anti-rabbit IgG (H+L) Ab, diluted at 1/600, was used as the secondary antibody.

Reference Citations:

1). Liu H et al. Hypoxia-inducible factor-1α promotes endometrial stromal cells migration and invasion by upregulating autophagy in endometriosis. Reproduction 2017 Jun;153(6):809-820 (PubMed: 28348069)

Application: WB 1/1000    Species: human;    Sample:Not available;

FIGURE 4. Knockdown of HIF-1α interferes with hypoxia-induced autophagy in HESCs. (A) Representative western blots of HIF-1α, Beclin1 and LC3 protein in HESCs transfected with scrambled control siRNA or HIF- 1α specific siRNA in the presence or absence of hypoxia. (B) The protein expression levels were quantified by Image J software and normalized to GAPDH protein levels. The data are presented as the means ± SD from at least three independent experiments (*p<0.05;**p<0.01; ***p

2). Xiang XJ et al. MiR-1271 Inhibits Cell Proliferation, Invasion and EMT in Gastric Cancer by Targeting FOXQ1. Cell Physiol Biochem 2015;36(4):1382-94 (PubMed: 26159618)

Application: WB    Species: human;    Sample: human gastric cancer;

Fig. 2. FOXQ1 is a direct target of miR-1271. (A) TargetScan (www.targetscan. org) and miRanda (www. showed that FOXQ1 was a direct target of miR-1271. (B) The luciferase reporter assay showed that FOXQ1 could be targeted by miR-1271 in MGC- 803 and SGC-7901 cells. (C) The qPCR and western blot analyses showed that miR-1271 mimics could inhibit the mRNA and protein expression of FOXQ1 in MGC-803 cells, whereas the miR-1271 inhibitor could increase the mRNA and protein expression of FOXQ1 in SGC-7901 cells. Three independent experiments were conducted. *P < 0.05, **P < 0.01.

3). Zhang J et al. Electrophysiological and trafficking defects of the SCN5A T353I mutation in Brugada syndrome are rescued by alpha-allocryptopine. Eur J Pharmacol 2015 Jan 5;746:333-43 (PubMed: 25261036)

Application: WB    Species:Not available;    Sample:Not available;

4). Sun H et al. Role of 5-HT1A receptor in insular cortex mediating stress - induced visceral sensory dysfunction. Neurogastroenterol Motil 2016 Jul;28(7):1104-13 (PubMed: 26969829)

Application: WB    Species:Not available;    Sample:Not available;

Figure 3 Comparison of the expression levels of HTR1As and NR2B in bilateral insular cortexes among three groups. The mRNA and protein expression levels of HTR1As (A and C) and NR2B (B and D) in bilateral insular cortexes were compared among WAS, sham WAS and normal control groups. (E) and (F): Representative immunoblots from the bilateral insular cortexes show the expression of NR2B, 5HTR1A and GAPDH, with an indication of size (kilodaltons, kDa). *represented that the difference was significant, p < 0.05. WAS, water avoidance stress.

5). Liu X et al. The role of Delta-like 4 ligand/Notch-ephrin-B2 cascade in the pathogenesis of preeclampsia by regulating functions of endothelial progenitor cell. Placenta 2015 Sep;36(9):1002-10 (PubMed: 26212082)

Application: WB 1/2000    Species: human;    Sample: human;

Fig. 2. Transfection and reagents influenced the expression of ephrin-B2 in EPCs.

6). Xiulong Niu et al. IL-17A exacerbates cisplatin-based resistance of OVCA via upregulating the expression of ABCG2 and MDR1 through Gli1-mediated Hh signaling. Oncotarget 2016

7). Gao T et al. Aberrant hypomethylation and overexpression of the eyes absent homologue 2 suppresses tumor cell growth of human lung adenocarcinoma cells. Oncol Rep 2015 Nov;34(5):2333-42 (PubMed: 26329363)

Application: WB    Species: human;    Sample: NSCLC cells;

Figure 1. Expression of EYA2 in NSCLC cells. (A) Western blot analysis. (B) EYA2 protein expression was significantly upregulated 3.2‑fold in the A549 cells compared to the BEAS-2B cells, but had no obvious change in the H661 and SKMES1 cells. (C) mRNA level of EYA2 exhibited a 2.1-fold upregulation in the A549 cells compared to the BEAS-2B cells. The groups were obtained from different parts of the same gel. * P

8). Zhao X et al. Up-regulation of miR-21 and miR-23a Contributes to As2 O3 -induced hERG Channel Deficiency. Basic Clin Pharmacol Toxicol 2015 Jun;116(6):516-23 (PubMed: 25395240)

Application: WB    Species:Not available;    Sample:Not available;

9). Fu WJ et al. Anti-atherosclerosis and cardio-protective effects of the Angong Niuhuang Pill on a high fat and vitamin D3 induced rodent model of atherosclerosis. J Ethnopharmacol 2017 Jan 4;195:118-126 (PubMed: 27880884)

10). Liu W et al. dNK derived IFN-γ mediates VSMC migration and apoptosis via the induction of LncRNA MEG3: A role in uterovascular transformation. Placenta 2017 Feb;50:32-39 (PubMed: 28161059)

11). Wang L et al. CDK3 is a major target of miR-150 in cell proliferation and anti-cancer effect. Exp Mol Pathol 2017 Apr;102(2):181-190 (PubMed: 28108217)

Application: WB 1/5000    Species: human;    Sample: H1299 cells;

Figure 6. miR-150 suppresses CDK3 expression in lung cancer compared cells. SPC1and H1299 cells were transfected with miR-150 mimics and mimics NC for forty-eight hours, respectively. A. Western blot of CDK3 in SPC1and H1299 cells. B. qRT-PCR analysis of miR-150 in SPC1and H1299 cells. C. qRT-PCR analysis of CDK3 mRNA in SPC1and H1299 cells. Data are the means ± SEM of three independent experiments. *p<0.05, **p

12). Mao X et al. Inhibitors of PARP-1 exert inhibitory effects on the biological characteristics of hepatocellular carcinoma cells in vitro. Mol Med Rep 2017 Jul;16(1):208-214 (PubMed: 28498459)

Application: WB 1/1000    Species: human;    Sample: HepG2;

Figure 4. Effects of different concentrations of AG014699 and BSI‑201 on protein levels of Caspase 3, Caspase 8, Bax and Bcl‑2 in HepG2 cells.(A) Blots showing proteins in cells treated with AGO14699 and (B) quantification. (C) Blots showing proteins in cells treated with (C) BSI‑201 and (D) quantification. * P<0.05, compared with the control group; ∆P<0.05, compared with the low dose group; ∆∆P<0.05, compared with the middle dose group. CTRL, control; Bcl‑2, B‑cell lymphoma 2; BAX, Bcl‑2‑associated X protein.

13). Liu X et al. Lipoxin A4 and its analog suppress inflammation by modulating HMGB1 translocation and expression in psoriasis. Sci Rep 2017 Aug 2;7(1):7100 (PubMed: 28769106)

Application: WB 1/1000    Species: human;    Sample:Not available;

Figure 5. LXA4 pretreatment downregulates the LPS-induced secretion and expression of HMGB1 in keratinocytes. NHEKs were stimulated with LPS (10μg/ml) with and without preincubation with LXA4 (100nmol/l) for 30minutes. (A) Western blot analysis shows specifc bands for the expression of nuclear and cytoplasmic HMGB1 in NHEKs. Full-length blots are presented in Suppl.

14). Zhang J et al. Long non-coding RNA NEAT1 regulates E2F3 expression by competitively binding to miR-377 in non-small cell lung cancer. Oncol Lett 2017 Oct;14(4):4983-4988 (PubMed: 29085511)

15). Liu YR et al. Twist1 confers multidrug resistance in colon cancer through upregulation of ATP-binding cassette transporters. Oncotarget 2017 May 2;8(32):52901-52912 (PubMed: 28881781)

16). Pan L et al. The cardiac glycoside oleandrin induces apoptosis in human colon cancer cells via the mitochondrial pathway. Cancer Chemother Pharmacol 2017 Jul;80(1):91-100 (PubMed: 28597038)

Application: WB 1/5000    Species: human;    Sample: SW480;

17). Wang X et al. Autophagy enhanced antitumor effect in K562 and K562/ADM cells using realgar transforming solution. Biomed Pharmacother 2018 Feb;98:252-264 (PubMed: 29272786)

18). Seo E et al. Exendin-4 in combination with adipose-derived stem cells promotes angiogenesis and improves diabetic wound healing. J Transl Med 2017 Feb 15;15(1):35 (PubMed: 28202074)

19). Tian Y et al. Magnolol Alleviates Inflammatory Responses and Lipid Accumulation by AMP-Activated Protein Kinase-Dependent Peroxisome Proliferator-Activated Receptor α Activation. Front Immunol 2018 Feb 5;9:147 (PubMed: 29467759)

20). Wu Y et al. Study on the mechanism of JAK2/STAT3 signaling pathway-mediated inflammatory reaction after cerebral ischemia. Mol Med Rep 2018 Apr;17(4):5007-5012 (PubMed: 29393445)

21). Seo E et al. Exendin-4 in combination with adipose-derived stem cells promotes angiogenesis and improves diabetic wound healing. J Transl Med 2017 Feb 15;15(1):35 (PubMed: 28202074)

22). Seo E et al. Exendin-4 in combination with adipose-derived stem cells promotes angiogenesis and improves diabetic wound healing. J Transl Med 2017 Feb 15;15(1):35 (PubMed: 28202074)

23). Yuan Y et al. Regulation of Signaling Pathways Involved in the Anti-proliferative and Apoptosis-inducing Effects of M22 against Non-small Cell Lung Adenocarcinoma A549 Cells. Sci Rep 2018 Jan 17;8(1):992 (PubMed: 29343765)

24). Seo E et al. Exendin-4 in combination with adipose-derived stem cells promotes angiogenesis and improves diabetic wound healing. J Transl Med 2017 Feb 15;15(1):35 (PubMed: 28202074)

25). Meng J et al. Twist1 Regulates Vimentin through Cul2 Circular RNA to Promote EMT in Hepatocellular Carcinoma. Cancer Res 2018 Aug 1;78(15):4150-4162 (PubMed: 29844124)

26). Li XH et al. Parthenolide attenuated bleomycin-induced pulmonary fibrosis via the NF-κB/Snail signaling pathway. Respir Res 2018 Jun 5;19(1):111 (PubMed: 29871641)

27). Liu H et al. Autophagy contributes to hypoxia-induced epithelial to mesenchymal transition of endometrial epithelial cells in endometriosis. Biol Reprod 2018 May 31 (PubMed: 29860279)

28). Hu J et al. Effects of Xin-Ji-Er-Kang on heart failure induced by myocardial infarction: Role of inflammation, oxidative stress and endothelial dysfunction. Phytomedicine 2018 Mar 15;42:245-257 (PubMed: 29655692)

29). Seo E et al. Exendin-4 in combination with adipose-derived stem cells promotes angiogenesis and improves diabetic wound healing. J Transl Med 2017 Feb 15;15(1):35 (PubMed: 28202074)

30). Ji S et al. Resveratrol promotes oxidative stress to drive DLC1 mediated cellular senescence in cancer cells. Exp Cell Res 2018 Sep 15;370(2):292-302 (PubMed: 29964052)

31). Seo E et al. Exendin-4 in combination with adipose-derived stem cells promotes angiogenesis and improves diabetic wound healing. J Transl Med 2017 Feb 15;15(1):35 (PubMed: 28202074)

32). Xu H et al. Involvement of insulin signalling pathway in methamphetamine-induced hyperphosphorylation of Tau. Toxicology 2018 Jul 4;408:88-94 (PubMed: 29981415)

33). Meng J et al. Hsp90β promotes aggressive vasculogenic mimicry via epithelial-mesenchymal transition in hepatocellular carcinoma. Oncogene 2018 Aug 7 (PubMed: 30087438)

34). Yun-Peng Shi et al. Synaptic plasticity of Human Umbilical Cord Mesenchymal Stem Cell differentiating into neuron like cells in vitro induced by Edavarone. STEM CELLS INT 2018 Aug;

35). Luo ML et al. Conditioned Medium from Human Umbilical Vein Endothelial Cells Promotes Proliferation, Migration, Invasion and Angiogenesis of Adipose Derived Stem Cells. Curr Med Sci 2018 Feb;38(1):124-130 (PubMed: 30074161)

36). Sun C et al. Hsa-miR-326 targets CCND1 and inhibits non-small cell lung cancer development. Oncotarget 2016 Feb 16;7(7):8341-59 (PubMed: 26840018)

Application: WB 1/5000    Species:Not available;    Sample:Not available;

Figure 8: Ectopic expression of miR-326 promotes apoptosis in A549 and SPC-A-1 cells. (C) Western-blot of Bcl2 protein in A549 and SPC-A-1 cells after transfection.

37). Zhan G et al. PGC-1α-FNDC5-BDNF signaling pathway in skeletal muscle confers resilience to stress in mice subjected to chronic social defeat. Psychopharmacology (Berl) 2018 Sep 26 (PubMed: 30259075)

38). Zhou J et al. Adipose derived mesenchymal stem cells alleviated osteoarthritis and chondrocyte apoptosis through autophagy inducing. J Cell Biochem 2018 Oct 13 (PubMed: 30315711)

39). Liu H et al. Long non-coding RNA MALAT1 mediates hypoxia-induced pro-survival autophagy of endometrial stromal cells in endometriosis. J Cell Mol Med 2018 Oct 15 (PubMed: 30324652)

40). Wang H et al. Oleanolic acid inhibits epithelial-mesenchymal transition of hepatocellular carcinoma by promoting iNOS dimerization. Mol Cancer Ther 2018 Oct 8 (PubMed: 30297361)

41). Liu SY et al. Fibroblast growth factor-23 may serve as a novel biomarker for renal osteodystrophy progression. Int J Mol Med 2018 Oct 15 (PubMed: 30365152)

42). Luo DD et al. Tetrahydrocurcumin and octahydrocurcumin, the primary and final hydrogenated metabolites of curcumin, possess superior hepatic-protective effect against acetaminophen-induced liver injury: Role of CYP2E1 and Keap1-Nrf2 pathway. Food Chem Toxicol 2018 Nov 10 (PubMed: 30423402)

43). Zhou Y et al. Magnesium ion leachables induce a conversion of contractile vascular smooth muscle cells to an inflammatory phenotype. J Biomed Mater Res B Appl Biomater 2018 Oct 1 (PubMed: 30270501)

44). Yangfei Xing et al. Dimerization of MICU Proteins Controls Ca 2 Influx Through the Mitochondrial Ca 2 Uniporter. SSRN Electronic Journal 2018

45). Wang KZ et al. Dietary gossypol reduced intestinal immunity and aggravated inflammation in on-growing grass carp (Ctenopharyngodon idella). Fish Shellfish Immunol 2018 Dec 10 (PubMed: 30543935)

46). Liu K et al. Melatonin reduces intramuscular fat deposition by promoting lipolysis and increasing mitochondrial function. J Lipid Res 2018 Dec 14 (PubMed: 30552289)

47). Hui Xiong et al. Tibetan medicine Kuan-Jin-Teng exerts anti-arthritic effects on collagen-induced arthritis rats via inhibition the production of pro-inflammatory cytokines and down-regulation of MAPK signaling pathway. Phytomedicine 2018

48). Chen L et al. Methamphetamine exposure induces neuropathic protein β-Amyloid expression. Toxicol In Vitro 2019 Feb;54:304-309 (PubMed: 30367931)

49). Zhao WX et al. Notch2 and Notch3 suppress the proliferation and mediate invasion of trophoblast cell lines. Biol Open 2017 Aug 15;6(8):1123-1129 (PubMed: 28606936)

50). Ren H et al. Endostatin attenuates PDGF-BB- or TGF-β1-induced HSCs activation via suppressing RhoA/ROCK1 signal pathways. Drug Des Devel Ther 2019 Jan 11;13:285-290 (PubMed: 30666090)

51). et al. Salidroside protects PC-12 cells against amyloid β-induced apoptosis by activation of the ERK1/2 and AKT signaling pathways.

52). Zhang Q et al. Thymidine phosphorylase promotes malignant progression in hepatocellular carcinoma through pentose Warburg effect. Cell Death Dis 2019 Jan 17;10(2):43 (PubMed: 30674871)

53). Xing Y et al. Dimerization of MICU Proteins Controls Ca2+ Influx through the Mitochondrial Ca2+ Uniporter. Cell Rep 2019 Jan 29;26(5):1203-1212 (PubMed: 30699349)

54). Chen C et al. A multifunctional-targeted nanoagent for dual-mode image-guided therapeutic effects on ovarian cancer cells. Int J Nanomedicine 2019 Jan 21;14:753-769 (PubMed: 30718954)

55). Li N et al. Notch activity mediates oestrogen-induced stromal cell invasion in endometriosis. Reproduction 2019 Feb 1 (PubMed: 30753135)

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Catalog Number :


Price/Size :

Tips: For phospho antibody, we provide phospho peptide(0.5mg) and non-phospho peptide(0.5mg).

Function :

Blocking peptides are peptides that bind specifically to the target antibody and block antibody binding. These peptide usually contains the epitope recognized by the antibody. Antibodies bound to the blocking peptide no longer bind to the epitope on the target protein. This mechanism is useful when non-specific binding is an issue, for example, in Western blotting (immunoblot) and immunohistochemistry (IHC). By comparing the staining from the blocked antibody versus the antibody alone, one can see which staining is specific; Specific binding will be absent from the western blot or immunostaining performed with the neutralized antibody.

Format and storage :

Synthetic peptide was lyophilized with 100% acetonitrile and is supplied as a powder. Reconstitute with 0.1 ml DI water for a final concentration of 1 mg/ml.The purity is >90%,tested by HPLC and MS.Storage Maintain refrigerated at 2-8°C for up to 6 months. For long term storage store at -20°C.

Precautions :

This product is for research use only. Not for use in diagnostic or therapeutic procedures.

IMPORTANT: For western blots, incubate membrane with diluted antibody in 5% w/v milk , 1X TBS, 0.1% Tween®20 at 4°C with gentle shaking, overnight.