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Human NAGase / N-Acetyl Beta-D-Glucosaminidase ELISA Kit

SKU:
HUFI02653
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P06865
Sensitivity:
0.938ng/ml
Range:
1.563-100ng/ml
ELISA Type:
Sandwich
Synonyms:
NAGase, N-Acetylhexosaminidase, beta-D-N-acetylhexosaminidase, beta-N-acetyl-D-hexosaminidase
Reactivity:
Human
€599
Frequently bought together:

Description

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Human NAGase/N-Acetyl Beta-D-Glucosaminidase ELISA Kit

The Human NAG (N-Acetyl-Beta-D-Glucosaminidase) ELISA Kit is specifically designed to accurately detect levels of NAG in human serum, plasma, and cell culture supernatants. This highly sensitive and specific kit ensures reliable and reproducible results, making it an excellent tool for a wide range of research applications.NAG is an important enzyme involved in the breakdown of complex carbohydrates, playing a key role in various cellular processes.

Abnormal levels of NAG have been associated with conditions such as kidney disease, liver disease, and metabolic disorders, making this kit a valuable resource for studying these conditions and potential therapeutic interventions.By using the Human NAG ELISA Kit, researchers can gain valuable insights into the role of NAG in health and disease, furthering our understanding of its impact on human biology.

Product Name:Human NAGase / N-Acetyl Beta-D-Glucosaminidase ELISA Kit
Product Code:HUFI02653
Size:96 Assays
Alias:NAGase, N-Acetylhexosaminidase, beta-D-N-acetylhexosaminidase, beta-N-acetyl-D-hexosaminidase
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human NAGase concentrations in serum plasma and other biological fluids.
Sensitivity:0.938ng/ml
Range:1.563-100ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human NAGase and the recovery rates were calculated by comparing the measured value to the expected amount of Human NAGase in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)87-10596
EDTA plasma(n=5)87-9892
UFH plasma(n=5)94-10497
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human NAGase and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)87-101%85-100%89-104%
EDTA plasma(n=5)93-101%86-101%82-91%
UFH plasma(n=5)85-96%88-99%80-88%
CV(%):Intra-Assay: CV<8%
Inter-Assay: CV<10%
ComponentQuantityStorage
ELISA Microplate (Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5-

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
UniprotP06865
UniProt Protein Function:HEXA: Responsible for the degradation of GM2 gangliosides, and a variety of other molecules containing terminal N-acetyl hexosamines, in the brain and other tissues. The form B is active against certain oligosaccharides. The form S has no measurable activity. Defects in HEXA are the cause of GM2-gangliosidosis type 1 (GM2G1); also known as Tay-Sachs disease. GM2- gangliosidosis is an autosomal recessive lysosomal storage disease marked by the accumulation of GM2 gangliosides in the neuronal cells. GM2G1 is characterized by GM2 gangliosides accumulation in the absence of HEXA activity, leading to neurodegeneration and, in the infantile form, death in early childhood. GM2G1 has an increased incidence among Ashkenazi Jews and French Canadians in eastern Quebec. It exists in several forms: infantile (most common and most severe), juvenile and adult (late onset). Belongs to the glycosyl hydrolase 20 family.
UniProt Protein Details:Protein type:Glycan Metabolism - glycosphingolipid biosynthesis - ganglio series; Glycan Metabolism - glycosaminoglycan degradation; Carbohydrate Metabolism - amino sugar and nucleotide sugar; Hydrolase; Glycan Metabolism - other glycan degradation; Glycan Metabolism - glycosphingolipid biosynthesis - globo series; EC 3.2.1.52

Chromosomal Location of Human Ortholog: 15q24.1

Cellular Component: lysosomal lumen; membrane

Molecular Function:protein heterodimerization activity; beta-N-acetylhexosaminidase activity

Biological Process: keratan sulfate metabolic process; chondroitin sulfate metabolic process; sphingolipid metabolic process; glycosaminoglycan metabolic process; chondroitin sulfate catabolic process; carbohydrate metabolic process; glycosphingolipid metabolic process; pathogenesis; keratan sulfate catabolic process; hyaluronan metabolic process; hyaluronan catabolic process

Disease: Tay-sachs Disease

NCBI Summary:This gene encodes the alpha subunit of the lysosomal enzyme beta-hexosaminidase that, together with the cofactor GM2 activator protein, catalyzes the degradation of the ganglioside GM2, and other molecules containing terminal N-acetyl hexosamines. Beta-hexosaminidase is composed of two subunits, alpha and beta, which are encoded by separate genes. Both beta-hexosaminidase alpha and beta subunits are members of family 20 of glycosyl hydrolases. Mutations in the alpha or beta subunit genes lead to an accumulation of GM2 ganglioside in neurons and neurodegenerative disorders termed the GM2 gangliosidoses. Alpha subunit gene mutations lead to Tay-Sachs disease (GM2-gangliosidosis type I). [provided by RefSeq, Jul 2009]
UniProt Code:P06865
NCBI GenInfo Identifier:311033393
NCBI Gene ID:3073
NCBI Accession:P06865.2
UniProt Secondary Accession:P06865,Q53HS8, Q6AI32, B4DKE7, E7ENH7,
UniProt Related Accession:P06865
Molecular Weight:529
NCBI Full Name:Beta-hexosaminidase subunit alpha
NCBI Synonym Full Names:hexosaminidase A (alpha polypeptide)
NCBI Official Symbol:HEXA  
NCBI Official Synonym Symbols:TSD  
NCBI Protein Information:beta-hexosaminidase subunit alpha; hexosaminidase subunit A; beta-N-acetylhexosaminidase subunit alpha; N-acetyl-beta-glucosaminidase subunit alpha
UniProt Protein Name:Beta-hexosaminidase subunit alpha
UniProt Synonym Protein Names:Beta-N-acetylhexosaminidase subunit alpha; Hexosaminidase subunit A; N-acetyl-beta-glucosaminidase subunit alpha
Protein Family:Hexamerin
UniProt Gene Name:HEXA  
UniProt Entry Name:HEXA_HUMAN

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

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

StepProtocol
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 °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 37°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 37°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 µl of TMB substrate into each well, cover the plate and incubate at 37°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 µl 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.

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 TypeProtocol
SerumIf 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.

PlasmaCollect 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 FluidCollect 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 supernatantCollect 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 lysatesSolubilize 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 homogenatesThe 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 lysatesRinse 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 MilkCollect 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.

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