Rat ALDH2 / Mitochondrial Aldehyde Dehydrogenase  ELISA Kit

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SKU:
RTFI00466
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P11884
Sensitivity:
0.188ng/ml
Range:
0.313-20ng/ml
ELISA Type:
Sandwich ELISA, Double Antibody
Synonyms:
ALDH2, Aldehyde dehydrogenase, mitochondrial, ALDHI, ALDH class 2, ALDH-E2, ALDM, Aldehyde Dehydrogenase 2
Reactivity:
Rat
Frequently bought together:

Description

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Product Name:Rat Aldh2 (Aldehyde dehydrogenase, mitochondrial) ELISA Kit
Product Code:RTFI00466
Size:96 Assays
Target:Rat Aldh2
Alias:ALDH2, Aldehyde dehydrogenase, mitochondrial, ALDHI, ALDH class 2, ALDH-E2, ALDM, Aldehyde Dehydrogenase 2
Reactivity:Rat
Detection Method:Sandwich ELISA, Double Antibody
Sensitivity:0.188ng/ml
Range:0.313-20ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Rat Aldh2 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat Aldh2 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)85-10194
EDTA plasma(n=5)88-10494
UFH plasma(n=5)90-10196
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat Aldh2 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)85-105%87-104%87-100%
EDTA plasma(n=5)82-100%82-94%85-101%
UFH plasma(n=5)82-100%80-95%80-96%
Intra-Assay:CV <8%
Inter-Assay:CV <10%
Uniprot:P11884
UniProt Protein Function:ALDH2: This protein belongs to the aldehyde dehydrogenase family of proteins. Aldehyde dehydrogenase is the second enzyme of the major oxidative pathway of alcohol metabolism. Two major liver isoforms of aldehyde dehydrogenase, cytosolic and mitochondrial, can be distinguished by their electrophoretic mobilities, kinetic properties, and subcellular localizations. Most Caucasians have two major isozymes, while approximately 50% of Orientals have the cytosolic isozyme but not the mitochondrial isozyme. A remarkably higher frequency of acute alcohol intoxication among Orientals than among Caucasians could be related to the absence of a catalytically active form of the mitochondrial isozyme. The increased exposure to acetaldehyde in individuals with the catalytically inactive form may also confer greater susceptibility to many types of cancer. This gene encodes a mitochondrial isoform, which has a low Km for acetaldehydes, and is localized in mitochondrial matrix. Alternative splicing results in multiple transcript variants encoding distinct isoforms.[provided by RefSeq, Mar 2011]
UniProt Protein Details:

Protein type:Amino Acid Metabolism - arginine and proline; Secondary Metabolites Metabolism - limonene and pinene degradation; Carbohydrate Metabolism - ascorbate and aldarate; Carbohydrate Metabolism - glycolysis and gluconeogenesis; Carbohydrate Metabolism - propanoate; Oxidoreductase; EC 1.2.1.3; Other Amino Acids Metabolism - beta-alanine; Carbohydrate Metabolism - pyruvate; Mitochondrial; Amino Acid Metabolism - lysine degradation; Amino Acid Metabolism - tryptophan; Amino Acid Metabolism - valine, leucine and isoleucine degradation; Amino Acid Metabolism - histidine; Lipid Metabolism - glycerolipid; Lipid Metabolism - fatty acid; Carbohydrate Metabolism - butanoate

Cellular Component: mitochondrial matrix; mitochondrion

Molecular Function:aldehyde dehydrogenase (NAD) activity; identical protein binding; oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor; protein binding

Biological Process: cellular response to hormone stimulus; ethanol catabolic process; liver development; negative regulation of apoptosis; response to estradiol stimulus; response to hyperoxia; response to lipopolysaccharide; response to nicotine; response to progesterone stimulus; response to testosterone stimulus

UniProt Code:P11884
NCBI GenInfo Identifier:14192933
NCBI Gene ID:29539
NCBI Accession:NP_115792.1
UniProt Secondary Accession:P11884,Q6Q288, Q6Q289, Q6Q290, Q8K3V8, Q91ZD7,
UniProt Related Accession:P11884
Molecular Weight:56,488 Da
NCBI Full Name:aldehyde dehydrogenase, mitochondrial
NCBI Synonym Full Names:aldehyde dehydrogenase 2 family (mitochondrial)
NCBI Official Symbol:Aldh2  
NCBI Protein Information:aldehyde dehydrogenase, mitochondrial
UniProt Protein Name:Aldehyde dehydrogenase, mitochondrial
UniProt Synonym Protein Names:ALDH class 2; ALDH-E2; ALDH1
Protein Family:NADP-dependent fatty aldehyde dehydrogenase
UniProt Gene Name:Aldh2  
UniProt Entry Name:ALDH2_RAT
StepProcedure
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 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 clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum 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 anti-coagulant. 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 & homogenizein 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.

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