Human Huntingtin (HTT) ELISA Kit

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SKU:
HUEB1201
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P42858
Range:
15.6-1000 pg/mL
ELISA Type:
Sandwich
Synonyms:
HTT, Huntingtin, Huntington disease protein, HD protein, HD, IT15
Reactivity:
Human
Frequently bought together:

Description

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Product Name:Human Huntingtin (HTT) ELISA Kit
Product Code:HUEB1201
Alias:Huntingtin, Huntington disease protein, HD protein, HTT, HD, IT15
Uniprot:P42858
Reactivity:Human
Range:15.6-1000 pg/mL
Detection Method:Sandwich
Size:96 Assay
Storage:Please see kit components below for exact storage details
Note:For research use only
UniProt Protein Function:Huntingtin: may play a role in microtubule-mediated transport or vesicle function. Widely expressed with the highest level of expression in the brain (nerve fibers, varicosities, and nerve endings). In the brain, the regions where it can be mainly found are the cerebellar cortex, the neocortex, the striatum, and the hippocampal formation. Defects are the cause of Huntington's disease, a neurodegenerative disorder characterized by loss of striatal neurons. This is thought to be caused by an expanded, unstable trinucleotide repeat in the Huntingtin gene, which translates as a polyglutamine repeat in the protein product. The Huntingtin locus is large, spanning 180 kb and consisting of 67 exons. The Huntingtin gene is widely expressed and is required for normal development. It is expressed as 2 alternatively polyadenylated isoforms displaying different relative abundance in various fetal and adult tissues.
UniProt Protein Details:

Protein type:Cytoskeletal

Chromosomal Location of Human Ortholog: 4p16.3

Cellular Component: nucleoplasm; Golgi apparatus; protein complex; cytoplasmic vesicle membrane; mitochondrion; axon; endoplasmic reticulum; late endosome; dendrite; cytoplasm; autophagic vacuole; inclusion body; cytosol; nucleus

Molecular Function:identical protein binding; protein binding; p53 binding; dynein intermediate chain binding; beta-tubulin binding; diazepam binding; transcription factor binding

Biological Process: ER to Golgi vesicle-mediated transport; citrulline metabolic process; paraxial mesoderm formation; regulation of protein phosphatase type 2A activity; regulation of synaptic plasticity; locomotory behavior; determination of adult life span; endosome transport; anterior/posterior pattern formation; L-glutamate import; regulation of mitochondrial membrane potential; establishment of mitotic spindle orientation; protein import into nucleus; organ development; quinolinate biosynthetic process; retrograde vesicle-mediated transport, Golgi to ER; vesicle transport along microtubule; visual learning; negative regulation of neuron apoptosis; Golgi organization and biogenesis; grooming behavior; endoplasmic reticulum organization and biogenesis; positive regulation of inositol-1,4,5-triphosphate receptor activity; striatum development; axon cargo transport; cell aging; olfactory lobe development; social behavior; lactate biosynthetic process from pyruvate; neuron apoptosis; iron ion homeostasis; insulin secretion; dopamine receptor signaling pathway; hormone metabolic process; neuron development; spermatogenesis; regulation of mitochondrial membrane permeability; response to calcium ion; neural plate formation; urea cycle

Disease: Huntington Disease

NCBI Summary:Huntingtin is a disease gene linked to Huntington's disease, a neurodegenerative disorder characterized by loss of striatal neurons. This is thought to be caused by an expanded, unstable trinucleotide repeat in the huntingtin gene, which translates as a polyglutamine repeat in the protein product. A fairly broad range in the number of trinucleotide repeats has been identified in normal controls, and repeat numbers in excess of 40 have been described as pathological. The huntingtin locus is large, spanning 180 kb and consisting of 67 exons. The huntingtin gene is widely expressed and is required for normal development. It is expressed as 2 alternatively polyadenylated forms displaying different relative abundance in various fetal and adult tissues. The larger transcript is approximately 13.7 kb and is expressed predominantly in adult and fetal brain whereas the smaller transcript of approximately 10.3 kb is more widely expressed. The genetic defect leading to Huntington's disease may not necessarily eliminate transcription, but may confer a new property on the mRNA or alter the function of the protein. One candidate is the huntingtin-associated protein-1, highly expressed in brain, which has increased affinity for huntingtin protein with expanded polyglutamine repeats. This gene contains an upstream open reading frame in the 5' UTR that inhibits expression of the huntingtin gene product through translational repression. [provided by RefSeq, Jul 2008]
UniProt Code:P42858
NCBI GenInfo Identifier:296434520
NCBI Gene ID:3064
NCBI Accession:P42858.2
UniProt Secondary Accession:P42858,Q9UQB7,
UniProt Related Accession:P42858
Molecular Weight:
NCBI Full Name:Huntingtin
NCBI Synonym Full Names:huntingtin
NCBI Official Symbol:HTT  
NCBI Official Synonym Symbols:HD; IT15  
NCBI Protein Information:huntingtin; huntington disease protein
UniProt Protein Name:Huntingtin
UniProt Synonym Protein Names:Huntington disease protein; HD protein
Protein Family:HD protein
UniProt Gene Name:HTT  
UniProt Entry Name:HD_HUMAN
Component Quantity (96 Assays) Storage
ELISA Microplate (Dismountable) 8×12 strips -20°C
Lyophilized Standard 2 -20°C
Sample Diluent 20ml -20°C
Assay Diluent A 10mL -20°C
Assay Diluent B 10mL -20°C
Detection Reagent A 120µL -20°C
Detection Reagent B 120µL -20°C
Wash Buffer 30mL 4°C
Substrate 10mL 4°C
Stop Solution 10mL 4°C
Plate Sealer 5 -

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

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

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1. Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2. Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3. Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4. Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5. Repeat the wash process for five times as conducted in step 3.
6. Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7. Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8. Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9. After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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 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.
Plasma 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.
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 & 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 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.
ELISA
Human HTT / Huntingtin ELISA Kit

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