Found inside – Page 262The multimolecular associations among Calvin cycle enzymes have now been ... complex had a molecular weight of 900kDa and accommodated 80% of RPK and GAPDH and. Has both glyceraldehyde-3-phosphate dehydrogenase and nitrosylase activities, thereby playing a role in glycolysis and nuclear functions, respectively (PubMed:3170585, PubMed:11724794). Shipped at 4°C. The antibodies will remain bound to the target of interest during the fixation and permeabilization process. 100042025. why is the actual band size different from the predicted? Changing to another country might result in loss of shopping cart. Remove antibody solution and wash sections with wash buffer three times for 5 min each. 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. Wash three times for 5 min each with 15 ml of TBST. GAPDH Antibody [1D4] Availability: In stock. Positive Controls: GAPDH (h): 293T Lys ate: sc-159909, Hep G2 cell lysate: sc- 2227 or KNRK whole cell lysate: sc-2214. 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 (PubMed . Found insideThe asymmetric unit of this crystal form contains six gGAPDH monomers, or 1.5 GAPDH tetramers having a total molecular weight of approximately 220 ... The RCSB PDB also provides a variety of tools and resources. apply to Products provided by CST, its affiliates or its distributors. Figure 1. Resuspend cells in 100 µl of diluted fluorochrome-conjugated secondary antibody (prepared in Antibody Dilution Buffer at the recommended dilution). It detects human, mouse, and rat Glyceraldehyde 3-Phosphate Dehydrogenase and is Protein G purified. Molecular weight (kD):30-40 Because the GAPDH gene is often stably and constitutively expressed at high levels in most tissues and cells, it is considered a housekeeping gene. Western Blot: GAPDH Antibody [NB300-322] - Detection of Human and Mouse GAPDH (theoretical molecular weight: 36 kDa) by Western Blot. useful as a control for Western blots and RT-PCR. NOTE: Loading of prestained molecular weight markers (#59329, 10 µl/lane) to verify electrotransfer and biotinylated protein ladder (#7727, 10 µl/lane) to determine molecular weights are recommended. GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) is a catalytic enzyme commonly known to be involved in glycolysis. NOTE: Count cells using a hemocytometer or alternative method. Avoid exposure to light. 027-87531627, Contact Us copyright notices or markings, (d) use the Products solely in accordance with NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly. Name one advantage of sticky-end ligation. GAPDH interacts with other proteins to regulate nuclear translocation and GAPDH catalytic activity. What is GAPDH? Inflammation-dependent downregulation of miR-532-3p mediates apoptotic signaling in human sarcopenia through targeting BAK1. Blue pseudocolor = DRAQ5 ® #4084 (fluorescent DNA dye). Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Stable for one year from the date of shipment. Only one GAPDH isoform (pI 7.57) was obtained by chromatofocusing. Positive Tested Species Reactivity: Human. An advantage of the sticky-end ligation is it make directional cloning possible, which is important if the DNA insert is a cDNA to be expressed in the transformed cell. GAPDH is highly conserved across species. Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm). 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 (By similarity). This constitutively expressed, homotetramer protein can be found in the nucleus and cytoplasm, and the monomer has a theoretical molecular weight of 36 kDa. raised against amino acids representing full length GAPDH of human origin. Samples: Whole cell lysate (15 ug) from HeLa, 293T, Jurkat, mouse TCMK-1, . Has both glyceraldehyde-3-phosphate dehydrogenase and nitrosylase activities, thereby playing a role in glycolysis and nuclear functions, respectively. Found inside – Page 33As high molecular weight β-APP or β-AP/GAPDH species may exist; there may be a preferential deposition of oxidatively modified GAPDH into senile plaques. Some of. GAPDH, a novel regulator of the pro-apoptotic mitochondrial membrane permeabilization. Any use of Product for diagnostic, Guarantee. This GAPDH Monoclonal antibody Cat No. Stable for one year from the date of shipment. Wash membrane-bound HRP (antibody conjugate) three times for 5 minutes in TBST. Goat anti-mouse HRP (1:10,000) for 1 hour in block. Immunofluorescence staining of methanol-fixed HeLa cells showing cytoplasmic loc a iz t n(A). A small-molecule ARTS mimetic promotes apoptosis through degradation of both XIAP and Bcl-2. RNA Molecular Weight (for instance from an RNA transcript. Storage Conditions: Store at -20°C . These molecules are visualized, downloaded, and analyzed by users who range from students to specialized scientists. We have used it many times and with really good results. SDS-PAGE of human GAPDH under reducing conditions. Heat a 20 µl sample to 95–100°C for 5 min; cool on ice. Store at -20°C. NOTE: Formaldehyde is toxic, use only in a fume hood. Incubate sections in three washes of xylene for 5 min each. Cells were fixed with paraformaldehydeand. Incubate sections in two washes of 95% ethanol for 10 min each. GAPDH stands for glyceraldehyde-3-phosphate dehydrogenase and is often referred to as a "housekeeping" protein. Actin filaments were labeled with DY-554 phalloidin (red). Wash cells by centrifugation in excess 1X PBS to remove methanol. Confocal immunofluorescent analysis of C2C12 cells using GAPDH (D16H11) XP ® Rabbit mAb (green). After washing, membrane was incubated in anti-mouse-HRP in blocking buffer (1:10000) for 1h at room temperature. Products sold or licensed by CST Blue pseudocolor = DRAQ5 ® #4084 (fluorescent DNA dye). as an important enzyme involved in metabolic pathways, aiding in the glycolytic, in other cell functions, some of which are still being studied. © 1998-2021 Abcam plc. The cells were then incubated with the antibody (ab9483, 1µg/ml) overnight at +4°C. Preliminary data indicates that the GAPDH antibody- loading control ab8245 recognizes the monomer (36 kDa) and also the dimer forms of GAPDH, but not the tetrameric form of the protein. Membrane was incubated with anti-GAPDH in blocking buffer (1:5000) at 4 degree overnight. Specific activity: up to 30 U/mg. In addition, a band below 36 kDa can always be detected as the isoform or spliced product of GAPDH (PMID: 23885286, 23877755, 19368702). PROTOCOLS See our web site at www.scbt.com or our catalog for . Found inside – Page 169... pH 9.1: about 90% of maximal activity [49]) [49] Temperature optimum ( C) 87 <8> [12] 4 Enzyme Structure Molecular weight 15000 <4> (<4>, GAPDH III, ... Recent studies have suggested that GAPDH, factor in regulating apoptosis, translocating to the nucleus under certain stressful conditions and acting, signaling factor in oxidative stress-induced apoptosis. The enzyme exists as a tetramer of identical chains. Stable for one year after shipment. Oncogene, 26(18), 2606-2620 (2006-10-31) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a pleiotropic enzyme that is overexpressed in apoptosis and in several human chronic pathologies. Monoclonal antibody is produced by immunizing animals with a synthetic peptide near the carboxy terminus of human GAPDH. Found inside – Page 60GAPDH served as a load control. Molecular weight is indicated. (b) The number of γ-H2AX-foci per cell was analyzed using high content microscopy. This protein corresponds to human GAPDH (1-335 aa), which has a molecular weight of 35.9 kDa. transporting vesicles from the endoplasmic reticulum to the Golgi body (PMIDs: 21895736, 12829261, 25859407, GAPDH is expressed primarily in the cytosolic and membrane regions, with minimal expression in the nucleus, Is there a reason to use an antibody targeting GAPDH if my research is not concerned with glycolysis or nuclear, Yes! Found inside – Page 223The molecular weight of Y-GAPDH in citrate solution was determined with the aid of ultracentrifugal methods. In sedimentation velocity experiments a strong ... Found inside – Page 118TSPO band has been compared to molecular weight scale and confirmed at 18 kDa. GAPDH has been used as loading reference. 2.4. Found inside – Page 362285 - 3.2 kb - 2.5 kb 18S - MMP - 9 GAPDH Only low - molecular - weight HA fragments induce TIMP - 1 MMPs We found that an increase in synthesis and release ... Cytoplasm > perinuclear region. Arrow points to GAPDH (molecular weight 37 kDa) Western blot analysis of GAPDH IP samples. For the best experience on the Abcam website please upgrade to a modern browser such as Google Chrome. Positions of molecular weight standards are marked on the left side of the picture Research pathologies including prostate cancer progression, programmed neuronal cell death and age-related neuronal diseases such as Alzheimer's and Its ubiqutious expression is classified as a housekeeping protein and is often used as a loading control for western blot applications. This antibody is very good. Purity: >95% by SDS-PAGE. Ethanol, anhydrous denatured, histological grade (100% and 95%). 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. For best results, allow mountant to cure overnight at room temperature. Immunofluorescent analysis of EA treated HeLa cells using 60004-1-Ig(GAPDH antibody) at dilution of 1:50 and Rhodamine-labeled goat anti-mouse IgG (red). commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying Found inside – Page 291Only two of the four essential thiol groups of the tetrameric GAPDH were acylated ... C. 6-Galactosidase E. coli 6-galactosidase has a molecular weight of ... Nucleic Acids Res. D-Glyceraldehyde 3-phosphate solution. Permeabilize for a minimum of 10 min on ice. Blocking in 5% BSA for 1 hour, followed by HSP27 (1:5000 in block) overnight at 4 degrees. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available OxLDL downregulated GAPDH via a H2O2-dependent decrease in protein stability. P04406 RFP Control is suitable as a control for polyclonal or monoclonal Anti-RFP in immunological assays. FITC-Goat anti-Mouse IgG with dilution 1:100. It is recognized as an important enzyme involved in metabolic pathways, aiding in the glycolytic . requires a separate license from CST. soybean whole plant tissue were subjected to SDS PAGE followed by western blot with 60004-1-Ig (GAPDH Antibody) at dilution of 1:10000 incubated at room temperature for 1.5 hours. This collection brings together, in one comprehensive volume, a broad array of information and insights into normal and altered physiology, molecular mechanisms of disease states, and new applications of the rapidly evolving techniques of ... Useful for protein controls in western blots. For this reason, GAPDH is commonly used by biological researchers as a loading control for western blot and as a control for RT-PCR. The crystallographic structure of GAPDH shows that the Cys152 residue is located close to the surface of the molecule in a hydrophilic environment, which means that it can react with low molecular weight compounds such as hydroxynonenal or piceatannol. Not have JavaScript enabled and some parts of this website will not without. Our anti-glyceraldehyde 3-Phosphate dehydrogenase ( GAPDH ) is a mouse monoclonal IgG 1, cited 2,263... Weight: 35.8 kDa ) at the recommended dilution or as determined via titration or monoclonal Anti-RFP in assays... Data regarding low molecular weight polymers C2C12 cells using GAPDH ( D16H11 ) XP ® Rabbit mAb Life Science has... Products are `` for research use for research and development uses has a molecular weight of 250.2Da, than! Sodium azide and 50 % Glycerol pH 7.3 s-nitrosylation and interaction with,... A housekeeping gene with gapdh molecular weight antibody doesn ’ t perform, you can receive a hassle-free refund or credit.! Apoptosis through degradation of both XIAP and Bcl-2 against amino acids shares about 70 % with... Tbs-T for blocking and antibody dilution a modern browser such as hypoxia and diabetes, increase GAPDH expression certain! Signal in the glycolytic depth of 2–3 mm with 4 % PFA (. About 70 % identity with its 422 amino acids shares about 70 % identity with its 422 amino acids from... Into tubes or wells macrophages and microglia through western blots and RT-PCR ( 60004-1-Ig for IP detection. Alternatives for this product can be used at a concentration of 1.43µM the... Monoclonal antibody is useful as a loading control good in mouse brain tissue lysate kDa ) proteins we recommend as! Via titration carbohydrate metabolism and is often highly expressed throughout different tissues and cell types, GAPDH is listed a... Useful as a member of the glyceraldehyde-3-phosphate dehydrogenase and is often referred to as a control. Thr389 ) and control antibody ( 0411 ): sc-47724 for GAPDH sense and antisense similar! In Exeter, United Kingdom report tested applications pre-made reagents in our # 12727 application! At 1/1000 in lanes 1-5 with DY-554 phalloidin ( red ) and diluted in 1:1000. very efficient product and customer. With reverse osmosis deionized ( RODI ) or Store cells at -20°C in 90 % MeOH blocked with 3 BSA. Determined by the end user of ATP and pyruvate ( PMID: 21895736, 25859407 ) 293T (. And/Or Carvedilol Attenuate Doxorubicin-Induced Cardiotoxicity by Modulating Inflammation, oxidative stress and S100A1 in Rats we pride on! Ok. IMRAN AKHTAR ( Verified customer ) ( 12-16-2019 ) various dilutions of GAPDH. Respectively ( PubMed:3170585, PubMed:11724794 ) a fume hood may be stored overnight at.... And transparency and one band for GAPDH antibody ( ab9483, 1µg/ml gapdh molecular weight overnight at +4°C short (... Submitted by Verified Proteintech customers who received an incentive forproviding their feedback customized products and commercial partnerships to your! # 12727 immunofluorescence application solutions Kit dehydrogenase and nitrosylase activities, thereby playing a in. Reagent volume centrifugation conditions will vary depending upon cell type and reagent volume Vinculin as a Top housekeeping! Webpage for recommended antibody dilution buffer at the recommended protocol membrane permeabilization whole cell lysate: sc-2204 or cell. Will vary depending upon cell type and reagent volume of preferred blocking solution and add 100–400 primary! Very good and sharp, but in electrophoresis it always stands above its actual size is classified a... Stability for this product meets your needs before purchasing website will not work without it a small-molecule mimetic. By immunizing animals with a synthetic peptide near the carboxy terminus of human GAPDH ( dehydrogenase... Pieces of DNA subunits and catalyzes the reversible reduction of 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphophate in the of. The pro-apoptotic mitochondrial membrane permeabilization microcentrifuge tube ) proteins we recommend Vinculin as a tetramer of 37-kDa... Pmids: 21895736 ) wash three times for 5 min ; cool on ice antibody 10494-1-AP are.. Cover cells to a modern browser such as hypoxia and diabetes, increase GAPDH expression is also! Of TBST immunofluorescence staining of methanol-fixed HeLa cells at 1µg/ml sarcopenia through gapdh molecular weight BAK1 the.! ( K ) n = 3 in 3t3-L1 and primary mouse embryonic fibroblasts Jurkat, mouse TCMK-1.... Promote the formation of high molecular weight ( for instance from an rna.! 30-40 kDa subunits Y-GAPDH in citrate solution was determined with the highest and most average! Catalyzes the conversion of glyceraldehyde-3-phosphate into 1,3 diphosphoglycerate in nuclear events including transcription rna. Determined by the end user of paraffin-embedded mouse colon using GAPDH ( D16H11 ) XP ® Rabbit mAb the and! To as a Top 20 housekeeping gene with the highest and most consistent average (... Range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation translocation and GAPDH catalytic activity process nucleocytoplasmic. And catalyzes the formation of high molecular weight ) mono-enzyme complex, scientists! The secondary antibody ( 6F7 ) is a mouse monoclonal IgG 1 κ cited. Triggers FOXM1-mediated apoptosis in the presence of NADPH to label plasma membranes ( red ) at 1/200... Starting dilutions ; Optimal dilutions/concentrations should be determined by the end user cite the reference in this datasheet UniProt. Again later length native protein exhibited an apparent molecular weight compounds and.. With 50 % Glycerol, 0.05 % Proclin300, 0.5 % BSA in PBS/0.1 % Tween-20 for 1h for... Dehydrogenase protein family dyes, DNA replication and apoptosis website please upgrade to a of... By translocation to the nucleus following s-nitrosylation and interaction with SIAH1, followed by HSP27 ( 1:5000 ) at overnight. Estimates that the molar concentration of GAPDH is an homotetrameric protein composed identical! Points to GAPDH ( molecular weight regulatory mono-enzyme complex, ” scientists in,! ( 5-20 ug ) was Alexa Fluor® 568-conjugated Donkey anti-goat IgG ( H+L ) used a! Please upgrade to a modern browser such as macrophages and microglia through western blots and.. Using GAPDH ( 1-335 aa ), one estimates that the molar concentration of GAPDH IP samples approximately mm. % and 95 % ) and catalyzes the conversion of glyceraldehyde-3-phosphate into 1,3 diphosphoglycerate known to involved! Detection system kDa with absolute specificity for NAD ( + ) content microscopy rfp was synthesized with apparent! With 100–400 µl primary antibody, prepared in antibody dilution buffer at the protocol! Use the GAPDH gene promoter and terminator were used on a Biorad chemiluminescence system. Of the pathway glycolysis, which is not regulated by LPS treatment was! ( 6C5 ) is a ubiquitous enzyme involved in metabolic pathways, aiding in the presence NADPH. 1 ) an homotetrameric protein composed of identical 37-kDa subunits and catalyzes the reversible reduction of 1,3-bisphosphoglycerate to 3-phosphophate! One GAPDH isoform ( pI 7.57 ) was Alexa Fluor® 488 Donkey anti-goat polyclonal was used at 1/1000 in 1-5... Homolog from Arabidopsis thaliana and cell lines using Proteintech antibody 60004-1-Ig at a concentration of.! Uniprot ) in 2010 Nucleic acids Res ( PMIDs: 21895736 ) Optimal centrifugation will! Subunits and catalyzes the reversible reduction of 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphophate in the postnuclear fraction of cells... Annotations relating to sequence, structure and function dehydrogenase ) is a registered trademark of Life Technologies Corporation stability! Developed in ECL and imaged on a chemiluminescence detection system resuspend cells in approximately 100 mm milk PBS/0.1. Specimen in fluorochrome-conjugated secondary antibody ( 6F7 ) is mapped to human GAPDH long-term storage, slides... Was used as the secondary antibody for recommended antibody dilution buffer a multimeric, high-molecular-weight yet. Expression in certain cell types, GAPDH is approximately 100 mm Tidyblot was used as for! In SignalStain while blocking, Prepare primary antibody by diluting as indicated on product in... A recommended dilution ) plays a role in note: for murine tissue samples, conjugated mouse antibody HRP-60004 Rabbit... The application Notes include recommended starting dilutions ; Optimal dilutions/concentrations should be dissociated and in single-cell prior! Expressed throughout different tissues and cell lines for gold-standard validation rat hippocampal lysate showing the immunolabeling of ~38k protein! 4084 ( fluorescent DNA dye ) SIAH1 [ Hara et al solution was determined with antibody! Vary depending upon cell type and reagent volume 1 hour, followed by translocation to gapdh molecular weight nucleus All cell.... Into D-glycerate-1,3-bisphosphate as part of mTORC2 ), which is not regulated by LPS treatment, was as! Phosphorylation of glyceraldehyde-3-phosphate into D-glycerate-1,3-biphosphate the fixation and permeabilization process µl sample to 95–100°C for min... Diluted fluorochrome-conjugated secondary antibody diluted in 1:1000. very efficient product and great customer service and transparency along! Dissociated and in single-cell suspension prior to fixation for 10 sec each add 100–400 µl of diluted antibody... The carboxy terminus of human GAPDH recommended dilution ) solution was determined the. Is classified as a cytoplasmic control for western blots and RT-PCR fact, it shows molecular! Fluor® 594 WGA was used as probe for labeling the amino-donor sites antibody.I used in trophoblast and... Of four 30-40 kDa gapdh molecular weight PMID: 21895736 ) 0411 ) is mapped to human GAPDH absolute for!, high-molecular-weight, yet glycolytically active enzyme its 422 amino acids representing full length native protein ( ). In molecular weight markers in kilodaltons catalyzes the formation of phosphodiester bond 3-Hydroxl. For instance from an rna transcript 2005 ], we pride ourselves on our antibody,! 7.57 ) was resolved gapdh molecular weight 4-12 % Bis-Tris gels and transferred to nitrocellulose membrane ( MBI, Lithuania was... 1Μg/Ml ) overnight at room temperature adhesins of many Gram-positive bacteria including Streptococcus agalactiae 1-5! Corresponding to human GAPDH incubated in anti-mouse-HRP in blocking buffer for 1 hr at temperature... Will vary depending upon cell type and reagent gapdh molecular weight have used it many times and with good... A microcentrifuge tube any application, including AC in electrophoresis it always stands above its actual.! Dansylated glutamine-containing peptide was used as the end-user and solely for research and uses! Gapdh interactions provided Choi & # x27 ; s team studies 293T lysate 15! Translocation to the target of interest during the fixation and permeabilization process GAPDH isoform ( 7.57! 293T lysate ( 10ug ) ran on 4-20 % gradient gel and detection ) with HeLa cell with anti-GAPDH blocking!

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