Human GAPDH ELISA Kit (HUEB2046)- High Sensitivity

Product Name:	Human Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ELISA Kit
SKU:	HUEB2046
Size:	96T
Target:	Human Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
Synonyms:	Peptidyl-cysteine S-nitrosylase GAPDH, CDABP0047, OK/SW-cl.12, GAPDH, GAPD
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.156-10ng/mL
Sensitivity:	0.1ng/mL

Intra CV:

4.7%

Inter CV:

7.8%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	98-108%	107-117%	100-110%	95-104%
EDTA Plasma(N=5)	107-115%	90-100%	91-100%	80-90%
Heparin Plasma(N=5)	95-106%	109-117%	101-110%	99-109%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	91	85-97
Plasma	93	87-99

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 (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.

Uniprot:	P04406
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Glyceraldehyde-3-phosphate dehydrogenase
Sub Unit:	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.
Research Area:	Neurosciences
Subcellular Location:	Cytoplasm Cytosol Nucleus Cytoplasm Perinuclear region Membrane Cytoplasm Cytoskeleton Translocates to the nucleus following S-nitrosylation and interaction with SIAH1, which contains a nuclear localization signal (By similarity). Postnuclear and Perinuclear regions.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	GAPDH: a multifunctional enzyme with both glyceraldehyde-3-phosphate dehydrogenase and nitrosylase activities. A key glycolytic enzyme that catalyzes the first step of the pathway by converting D-glyceraldehyde 3-phosphate (G3P) into 3-phospho-D-glyceroyl phosphate. An important enzyme for energy metabolism, and the production of ATP and pyruvate through anaerobic glycolysis in the cytoplasm. Additionally, it participates in apoptosis, membrane trafficking, iron metabolism, nuclear activities and receptor mediated cell signaling. Its subcellular localization changes reflecting its multiple activities. Is cytosolic, but is also localized in the membrane, the nucleus, polysomes, the ER and the Golgi. Participates in transcription, RNA transport, DNA replication and apoptosis. S-nitrosylation on Cys-152 following apoptotic stimulates its interaction with SIAH2, which in turn moderates its translocation into the nucleus. Mediates cysteine S-nitrosylation of nuclear target proteins including SIRT1, HDAC2 and DNA-PK. Deregulated in lung cancer, renal cancer, breast cancer, gastric cancer, glioma, liver cancer, colorectal cancer, melanoma, prostatic cancer, pancreatic cancer and bladder cancer. Its increased expression and enzymatic activity is associated with cell proliferation and tumorigenesis, Oxidative stress impairs GAPDH catalytic activity and leads to cellular aging and apoptosis. In experimental animal models, injection of GAPDH antagonists induces apoptosis and blocks Hep3B tumor progression, suggesting a therapeutic potential of targeting GAPDH in human hepatocellular carcinoma
UniProt Protein Details:	Protein type:Oxidoreductase; EC 1.2.1.12; Carbohydrate Metabolism - glycolysis and gluconeogenesis Chromosomal Location of Human Ortholog: 12p13 Cellular Component: microtubule cytoskeleton; nuclear membrane; membrane; intracellular membrane-bound organelle; perinuclear region of cytoplasm; cytoplasm; plasma membrane; lipid particle; cytosol; ribonucleoprotein complex; nucleus; vesicle Molecular Function:identical protein binding; protein binding; microtubule binding; glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) activity; NADP binding; NAD binding Biological Process: neuron apoptosis; glycolysis; protein stabilization; negative regulation of translation; carbohydrate metabolic process; glucose metabolic process; pathogenesis; microtubule cytoskeleton organization and biogenesis; gluconeogenesis
NCBI Summary:	This gene encodes a member of the glyceraldehyde-3-phosphate dehydrogenase protein family. The encoded protein has been identified as a moonlighting protein based on its ability to perform mechanistically distinct functions. The product of this gene catalyzes an important energy-yielding step in carbohydrate metabolism, the reversible oxidative phosphorylation of glyceraldehyde-3-phosphate in the presence of inorganic phosphate and nicotinamide adenine dinucleotide (NAD). The encoded protein has additionally been identified to have uracil DNA glycosylase activity in the nucleus. Also, this protein contains a peptide that has antimicrobial activity against E. coli, P. aeruginosa, and C. albicans. Studies of a similar protein in mouse have assigned a variety of additional functions including nitrosylation of nuclear proteins, the regulation of mRNA stability, and acting as a transferrin receptor on the cell surface of macrophage. Many pseudogenes similar to this locus are present in the human genome. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Nov 2014]
UniProt Code:	P04406
NCBI GenInfo Identifier:	120649
NCBI Gene ID:	2597
NCBI Accession:	P04406.3
UniProt Secondary Accession:	P04406,P00354, Q53X65, E7EUT4,
UniProt Related Accession:	P04406
Molecular Weight:	Calculated MW: 31kDa/36kDaObserved MW: 36kDa
NCBI Full Name:	Glyceraldehyde-3-phosphate dehydrogenase
NCBI Synonym Full Names:	glyceraldehyde-3-phosphate dehydrogenase
NCBI Official Symbol:	GAPDH
NCBI Official Synonym Symbols:	G3PD; GAPD; HEL-S-162eP
NCBI Protein Information:	glyceraldehyde-3-phosphate dehydrogenase; aging-associated gene 9 protein; peptidyl-cysteine S-nitrosylase GAPDH; epididymis secretory sperm binding protein Li 162eP
UniProt Protein Name:	Glyceraldehyde-3-phosphate dehydrogenase
UniProt Synonym Protein Names:	Peptidyl-cysteine S-nitrosylase GAPDH (EC:2.6.99.-)
UniProt Gene Name:	GAPDH
UniProt Entry Name:	G3P_HUMAN

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.

ELISA

Human GAPDH ELISA Kit