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Human HARS / Histidyl-tRNA synthetase, cytoplasmic ELISA Kit

SKU:
HUFI01403
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P12081
Sensitivity:
0.094ng/ml
Range:
0.156-10ng/ml
ELISA Type:
Sandwich
Synonyms:
HARS, Histidyl-tRNA synthetase, cytoplasmic, Histidine--tRNA ligase, HisRS, HRS
Reactivity:
Human
Research Area:
Metabolism
€599
Frequently bought together:

Description

Human HARS / Histidyl-tRNA synthetase, cytoplasmic ELISA

HARS1 (Histidyl-TRNA Synthetase 1) is a Protein Coding gene. GO annotations related to HARS1 (Histidyl-TRNA Synthetase 1) include aminomethyltransferase activity and transferase activity, transferring nitrogenous groups. HARS1 (Histidyl-TRNA Synthetase 1) has been known for harboring mutation causing MDS/AML. HARS1 encodes a nuclear protein that is involved in tRNA biosynthesis, which are essential for the translation process that helps produce proteins from RNA code. Few related pathways to HARS1 are Metabolism and O-phosphoserine that is converted into phosphoethanolamine by the HARS1/PHE1 complex.

system_update_altDatasheetsystem_update_altMSDS
Product Name:Human HARS / Histidyl-tRNA synthetase, cytoplasmic ELISA Kit
Product Code:HUFI01403
Size:96 Assays
Alias:HARS, Histidyl-tRNA synthetase, cytoplasmic, Histidine--tRNA ligase, HisRS, HRS
Detection method:Sandwich ELISA, Double Antibody
Application:This immunoassay kit allows for the in vitro quantitative determination of Human HARS concentrations in serum plasma and other biological fluids.
Sensitivity:0.094ng/ml
Range:0.156-10ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Human HARS and the recovery rates were calculated by comparing the measured value to the expected amount of Human HARS in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)90-10497
EDTA plasma(n=5)87-10596
UFH plasma(n=5)85-10493
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human HARS and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)89-105%89-99%85-98%
EDTA plasma(n=5)87-100%84-94%83-96%
UFH plasma(n=5)80-99%84-99%81-94%
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
UniprotP12081
UniProt Protein Function:HARS: Defects in HARS are a cause of Usher syndrome type 3B (USH3B). USH3B is a syndrome characterized by progressive vision and hearing loss during early childhood. Some patients have the so-called °Charles Bonnet syndrome,' involving decreased visual acuity and vivid visual hallucinations. USH is a genetically heterogeneous condition characterized by the association of retinitis pigmentosa with sensorineural deafness. Age at onset and differences in auditory and vestibular function distinguish Usher syndrome type 1 (USH1), Usher syndrome type 2 (USH2) and Usher syndrome type 3 (USH3). USH3 is characterized by postlingual, progressive hearing loss, variable vestibular dysfunction, and onset of retinitis pigmentosa symptoms, including nyctalopia, constriction of the visual fields, and loss of central visual acuity, usually by the second decade of life. Belongs to the class-II aminoacyl-tRNA synthetase family.
UniProt Protein Details:Protein type:EC 6.1.1.21; Translation; Ligase

Chromosomal Location of Human Ortholog: 5q31.3

Cellular Component: cytoplasm; cytosol; mitochondrion

Molecular Function:histidine-tRNA ligase activity

Biological Process: histidyl-tRNA aminoacylation; mitochondrial translation; tRNA aminoacylation for protein translation

Disease: Charcot-marie-tooth Disease, Axonal, Type 2w; Usher Syndrome, Type Iiib

NCBI Summary:Aminoacyl-tRNA synthetases are a class of enzymes that charge tRNAs with their cognate amino acids. The protein encoded by this gene is a cytoplasmic enzyme which belongs to the class II family of aminoacyl-tRNA synthetases. The enzyme is responsible for the synthesis of histidyl-transfer RNA, which is essential for the incorporation of histidine into proteins. The gene is located in a head-to-head orientation with HARSL on chromosome five, where the homologous genes share a bidirectional promoter. The gene product is a frequent target of autoantibodies in the human autoimmune disease polymyositis/dermatomyositis. Several transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Apr 2012]
UniProt Code:P12081
NCBI GenInfo Identifier:135123
NCBI Gene ID:3035
NCBI Accession:P12081.2
UniProt Secondary Accession:P12081,B4DHQ1, B4DY73, D6REN6, J3KNE5,
UniProt Related Accession:P12081
Molecular Weight:54,847 Da
NCBI Full Name:Histidine--tRNA ligase, cytoplasmic
NCBI Synonym Full Names:histidyl-tRNA synthetase
NCBI Official Symbol:HARS
NCBI Official Synonym Symbols:HRS; CMT2W; USH3B
NCBI Protein Information:histidine--tRNA ligase, cytoplasmic
UniProt Protein Name:Histidine--tRNA ligase, cytoplasmic
UniProt Synonym Protein Names:Histidyl-tRNA synthetase; HisRS
Protein Family:Histidine--tRNA ligase
UniProt Gene Name:HARS
UniProt Entry Name:SYHC_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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