Mouse GAPDH ELISA Kit
Mouse GAPDH ELISA Kit - Information
The ELISA Genie Mouse GAPDH ELISA Kit can assay for Mouse GAPDH in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.How our Mouse GAPDH ELISA Kits Work?
The ELISA Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, ELISA Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.
At ELISA Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound Mouse GAPDH is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.
Mouse GAPDH ELISA Kit - Data
Has both glyceraldehyde-3-phosphate dehydrogenase and nitrosylase activities, thereby playing a role in glycolysis and nuclear functions, respectively. 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. 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. Also 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. 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 Modification|| |
ISGylated. S-nitrosylation of Cys-150 leads to interaction with SIAH1, followed by translocation to the nucleus S-nitrosylation of Cys-245 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. Sulfhydration at Cys-150 increases catalytic activity. Oxidative stress can promote the formation of high molecular weight disulfide-linked GAPDH aggregates, through a process called nucleocytoplasmic coagulation.
|Detection method|| |
Sandwich ELISA Double Antibody
This immunoassay kit allows for the in vitro quantitative determination of Gapdh/Peptidyl-cysteine S-nitrosylase GAPDH/GAPD/G3PD/G3PDH/aging-associated gene 9 protein concentrations in serum plasma and other biological fluids.
4'C for 6 months
Matrices listed below were spiked with certain level of Gapdh/Peptidyl-cysteine S-nitrosylase GAPDH/GAPD/G3PD/G3PDH/aging-associated gene 9 protein and the recovery rates were calculated by comparing the measured value to the expected amount of Gapdh/Peptidyl-cysteine S-nitrosylase GAPDH/GAPD/G3PD/G3PDH/aging-associated gene 9 protein in samples.
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Gapdh/Peptidyl-cysteine S-nitrosylase GAPDH/GAPD/G3PD/G3PDH/aging-associated gene 9 protein and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
For Research Use Only
Mouse GAPDH ELISA Kit Protocol
The below protocol is a sample protocol for Mouse GAPDH ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Mouse GAPDH present in their sample.
Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37 Â°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.
|1.||Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!|
|2.||Aliquot 0.1ml standard solutions into the standard wells.|
|3.||Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.|
|4.||Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.|
|5.||Seal the plate with a cover and incubate at 37 Â°C for 90 min.|
|6.||Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.|
|7.||Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.|
|8.||Seal the plate with a cover and incubate at 37Â°C for 60 min.|
|9.||Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.|
|10.||Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37Â°C for 30 min.|
|11.||Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.|
|12.||Add 90 Âµl of TMB substrate into each well, cover the plate and incubate at 37Â°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.|
|13.||Add 50 Âµl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.|
|14.||Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.|
Mouse GAPDH ELISA Kit components
|ELISA Microplate(Dismountable)||8×12 strips||4°C for 6 months|
|Sample/Standard Dilution Buffer||20ml||4°C|
|Biotin-labeled Antibody(Concentrated)||120ul||4°C (Protect from light)|
|Antibody Dilution Buffer||10ml||4°C|
|HRP-Streptavidin Conjugate(SABC)||120ul||4°C (Protect from light)|
|SABC Dilution Buffer||10ml||4°C|
|TMB Substrate||10ml||4°C (Protect from light)|
Other materials and equipment required:The ELISA Genie Mouse GAPDH ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
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.
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.
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.
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.
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.
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.
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.
Mouse GAPDH ELISA Kit Protein Information
|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:EC 188.8.131.52; Oxidoreductase; Carbohydrate Metabolism - glycolysis and gluconeogenesis
Cellular Component: cytoplasm; cytosol; intracellular membrane-bound organelle; lipid particle; membrane; microtubule cytoskeleton; mitochondrion; nuclear membrane; nucleus; plasma membrane; ribonucleoprotein complex; vesicle
Molecular Function:glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) activity; identical protein binding; microtubule binding
Biological Process: apoptosis; carbohydrate metabolic process; gluconeogenesis; glycolysis; microtubule cytoskeleton organization and biogenesis; multicellular organismal development; negative regulation of translation; neuron apoptosis; protein stabilization
|NCBI GenInfo Identifier:||120702|
|NCBI Gene ID:||100042025|
|UniProt Secondary Accession:||P16858,Q0QEU0, Q3THM2, Q3TUI2, Q3UMT2, Q4V783, Q569X2 Q569X5, Q5U410, A6H6A8,|
|Molecular Weight:||35,810 Da|
|NCBI Full Name:||Glyceraldehyde-3-phosphate dehydrogenase|
|NCBI Synonym Full Names:||glyceraldehyde-3-phosphate dehydrogenase, pseudogene 15|
|NCBI Official Symbol:||Gapdh-ps15|
|NCBI Official Synonym Symbols:||Gm20899|
|NCBI Protein Information:||glyceraldehyde-3-phosphate dehydrogenase, pseudogene 15|
|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_MOUSE|