Human Reelin (RELN) ELISA Kit (HUEB2200)- High Sensitivity

Product Name:	Human Reelin (RELN) ELISA Kit
SKU:	HUEB2200
Size:	96T
Target:	Human Reelin (RELN)
Synonyms:	Reelin, RELN, 3.4.21.-
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	15.6-1000pg/mL
Sensitivity:	8.5pg/mL

Intra CV:

4.7%

Inter CV:

7.7%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	85-95%	102-110%	85-95%	92-103%
EDTA Plasma(N=5)	106-117%	102-114%	83-92%	102-113%
Heparin Plasma(N=5)	103-111%	111-120%	94-104%	80-90%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	95	89-101
Plasma	97	91-103

Function:

Extracellular matrix serine protease that plays a role in layering of neurons in the cerebral cortex and cerebellum. Regulates microtubule function in neurons and neuronal migration. Affects migration of sympathetic preganglionic neurons in the spinal cord, where it seems to act as a barrier to neuronal migration. Enzymatic activity is important for the modulation of cell adhesion. Binding to the extracellular domains of lipoprotein receptors VLDLR and LRP8/APOER2 induces tyrosine phosphorylation of DAB1 and modulation of TAU phosphorylation.

Uniprot:	P78509
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Reelin
Sub Unit:	Oligomer of disulfide-linked homodimers. Binds to the ectodomains of VLDLR and LRP8/APOER2.
Research Area:	Neurosciences
Subcellular Location:	Secreted Extracellular space Extracellular matrix
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	reelin: Extracellular matrix serine protease that plays a role in layering of neurons in the cerebral cortex and cerebellum. Regulates microtubule function in neurons and neuronal migration. Affects migration of sympathetic preganglionic neurons in the spinal cord, where it seems to act as a barrier to neuronal migration. Enzymatic activity is important for the modulation of cell adhesion. Binding to the extracellular domains of lipoprotein receptors VLDLR and LRP8/APOER2 induces tyrosine phosphorylation of DAB1 and modulation of TAU phosphorylation. Defects in RELN are the cause of lissencephaly type 2 (LIS2); also known as lissencephaly with cerebellar hypoplasia or Norman-Roberts syndrome. LIS2 is a classic type lissencephaly associated with abnormalities of the cerebellum, hippocampus and brainstem. Individuals with LIS2 are severely ataxic, mentally retarded and suffer from epilepsy. Belongs to the reelin family. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Cell development/differentiation; Secreted; Cell adhesion; EC 3.4.21.-; Protease; Secreted, signal peptide; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 7q22 Cellular Component: extracellular space; proteinaceous extracellular matrix; cytoplasm; dendrite Molecular Function:serine-type peptidase activity; metal ion binding; protein serine/threonine/tyrosine kinase activity Biological Process: cellular morphogenesis during differentiation; axon guidance; central nervous system development; peptidyl-tyrosine phosphorylation; spinal cord patterning; neuron migration; response to pain; proteolysis; positive regulation of synaptic transmission, glutamatergic; glial cell differentiation; dendrite development; cell adhesion; layer formation in the cerebral cortex; positive regulation of TOR signaling pathway; associative learning; regulation of behavior; regulation of synaptic transmission; cerebral cortex tangential migration; hippocampus development; activation of CREB transcription factor; ventral spinal cord development; positive regulation of phosphoinositide 3-kinase cascade; positive regulation of small GTPase mediated signal transduction; positive regulation of peptidyl-tyrosine phosphorylation; long-term memory; positive regulation of protein kinase activity; brain development Disease: Epilepsy, Familial Temporal Lobe, 7; Lissencephaly 2; Epilepsy, Familial Temporal Lobe, 1
NCBI Summary:	This gene encodes a large secreted extracellular matrix protein thought to control cell-cell interactions critical for cell positioning and neuronal migration during brain development. This protein may be involved in schizophrenia, autism, bipolar disorder, major depression and in migration defects associated with temporal lobe epilepsy. Mutations of this gene are associated with autosomal recessive lissencephaly with cerebellar hypoplasia. Two transcript variants encoding distinct isoforms have been identified for this gene. Other transcript variants have been described but their full length nature has not been determined. [provided by RefSeq, Jul 2008]
UniProt Code:	P78509
NCBI GenInfo Identifier:	1743885
NCBI Gene ID:	5649
NCBI Accession:	AAC51105.1
UniProt Secondary Accession:	P78509,Q86UJ0, Q86UJ8, Q8NDV0, Q9UDQ2, A4D0P9, A4D0Q0
UniProt Related Accession:	P78509
Molecular Weight:	384,093 Da
NCBI Full Name:	reelin
NCBI Synonym Full Names:	reelin
NCBI Official Symbol:	RELN
NCBI Official Synonym Symbols:	RL; ETL7; LIS2; PRO1598
NCBI Protein Information:	reelin
UniProt Protein Name:	Reelin
UniProt Gene Name:	RELN
UniProt Entry Name:	RELN_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 Reelin ELISA Kit