Human ACAT1 / Acetyl-CoA acetyltransferase ELISA Kit

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SKU:
HUFI00581
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P24752
Sensitivity:
4.688pg/ml
Range:
7.813-500pg/ml
ELISA Type:
Sandwich ELISA, Double Antibody
Synonyms:
ACAT1, AACAT, MAT, Acetyl-CoA acetyltransferase, mitochondrial, Acetoacetyl-CoA thiolase, T2
Reactivity:
Human
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Description

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Product Name:Human ACAT1 / Acetyl-CoA acetyltransferase ELISA Kit
Product Code:HUFI00581
Size:96 Assays
Alias:ACAT1, AACAT, MAT, Acetyl-CoA acetyltransferase, mitochondrial, Acetoacetyl-CoA thiolase, T2
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human ACAT1 concentrations in serum plasma and other biological fluids.
Sensitivity:4.688pg/ml
Range:7.813-500pg/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human ACAT1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human ACAT1 in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)90-10599
EDTA plasma(n=5)86-10599
UFH plasma(n=5)86-9491
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human ACAT1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)85-98%86-100%86-102%
EDTA plasma(n=5)82-97%83-94%89-99%
UFH plasma(n=5)89-100%86-100%87-100%
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
UniprotP24752
UniProt Protein Function:ACAT1: Plays a major role in ketone body metabolism. Defects in ACAT1 are a cause of 3-ketothiolase deficiency (3KTD); also known as alpha- methylacetoaceticaciduria. 3KTD is an inborn error of isoleucine catabolism characterized by intermittent ketoacidotic attacks associated with unconsciousness. Some patients die during an attack or are mentally retarded. Urinary excretion of 2-methyl-3- hydroxybutyric acid, 2-methylacetoacetic acid, triglylglycine, butanone is increased. It seems likely that the severity of this disease correlates better with the environmental or acquired factors than with the ACAT1 genotype. Belongs to the thiolase family.
UniProt Protein Details:Protein type:Secondary Metabolites Metabolism - terpenoid backbone biosynthesis; Amino Acid Metabolism - valine, leucine and isoleucine degradation; Lipid Metabolism - fatty acid; Acetyltransferase; Lipid Metabolism - synthesis and degradation of ketone bodies; Carbohydrate Metabolism - propanoate; Amino Acid Metabolism - tryptophan; EC 2.3.1.9; Amino Acid Metabolism - lysine degradation; Carbohydrate Metabolism - pyruvate; Carbohydrate Metabolism - butanoate; Mitochondrial

Chromosomal Location of Human Ortholog: 11q22.3

Cellular Component: mitochondrial matrix; mitochondrion

Molecular Function:acetyl-CoA C-acetyltransferase activity

Biological Process: branched chain family amino acid catabolic process; ketone body biosynthetic process; ketone body catabolic process

Disease: Alpha-methylacetoacetic Aciduria

NCBI Summary:This gene encodes a mitochondrially localized enzyme that catalyzes the reversible formation of acetoacetyl-CoA from two molecules of acetyl-CoA. Defects in this gene are associated with 3-ketothiolase deficiency, an inborn error of isoleucine catabolism characterized by urinary excretion of 2-methyl-3-hydroxybutyric acid, 2-methylacetoacetic acid, tiglylglycine, and butanone. [provided by RefSeq, Feb 2009]
UniProt Code:P24752
NCBI GenInfo Identifier:135755
NCBI Gene ID:38
NCBI Accession:P24752.1
UniProt Secondary Accession:P24752,Q96FG8, B2R6H1, G3XAB4,
UniProt Related Accession:P24752
Molecular Weight:17,175 Da
NCBI Full Name:Acetyl-CoA acetyltransferase, mitochondrial
NCBI Synonym Full Names:acetyl-CoA acetyltransferase 1
NCBI Official Symbol:ACAT1
NCBI Official Synonym Symbols:T2; MAT; ACAT; THIL
NCBI Protein Information:acetyl-CoA acetyltransferase, mitochondrial
UniProt Protein Name:Acetyl-CoA acetyltransferase, mitochondrial
UniProt Synonym Protein Names:Acetoacetyl-CoA thiolase; T2
UniProt Gene Name:ACAT1
UniProt Entry Name:THIL_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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