Rat Agrin ELISA Kit (RTFI00560)- High Sensitivity

Product Name:	Rat AGRN (Agrin) ELISA Kit
Product Code:	RTFI00560
Size:	96 Assays
Target:	Rat AGRN
Alias:	AGRN, Agrin, agrin proteoglycan
Reactivity:	Rat
Detection Method:	Sandwich ELISA, Double Antibody
Sensitivity:	18.75pg/ml
Range:	31.25-2000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Rat AGRN and the recovery rates were calculated by comparing the measured value to the expected amount of Rat AGRN in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-101	95
EDTA plasma(n=5)	85-100	94
UFH plasma(n=5)	87-101	93

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat AGRN and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	89-104%	85-103%	98-102%
EDTA plasma(n=5)	85-100%	86-98%	83-98%
UFH plasma(n=5)	83-92%	84-100%	81-98%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

P25304

UniProt Protein Function:	AGRN: Plays a central role in the formation and the maintenance of the neuromuscular junction (NMJ), the synapse between motor neuron and skeletal muscle. Ligand of the MUSK signaling complex that directly binds LRP4 in this complex and induces the phosphorylation of MUSK, the kinase of the complex. The activation of MUSK in myotubes induces the formation of NMJ by regulating different processes including the transcription of specific genes and the clustering of AChR in the postsynaptic membrane. Defects in AGRN are a cause of myasthenia, limb-girdle, familial (LGM). A congenital myasthenic syndrome characterized by a typical 'limb girdle' pattern of muscle weakness with small, simplified neuromuscular junctions but normal acetylcholine receptor and acetylcholinesterase function.
UniProt Protein Details:	Protein type:Apoptosis; Cell adhesion; Cell development/differentiation; Extracellular matrix; Cell surface; Secreted, signal peptide; Secreted Cellular Component: basal lamina; basement membrane; cell junction; cell surface; cytoplasm; extracellular matrix; extracellular region; extracellular space; integral to membrane; plasma membrane; proteinaceous extracellular matrix; sarcolemma; synapse Molecular Function:acetylcholine receptor regulator activity; ATPase inhibitor activity; calcium ion binding; heparan sulfate proteoglycan binding; laminin binding; protein binding; sialic acid binding; transforming growth factor beta binding Biological Process: acetylcholine receptor signaling, muscarinic pathway; neuromuscular junction development; neurotransmitter receptor metabolic process; plasma membrane organization and biogenesis; positive regulation of filopodium formation; positive regulation of GTPase activity; positive regulation of neuron apoptosis; positive regulation of peptidyl-tyrosine phosphorylation; positive regulation of protein amino acid phosphorylation; positive regulation of protein binding; positive regulation of synaptic growth at neuromuscular junction; positive regulation of transcription from RNA polymerase II promoter; protein heterotetramerization; receptor clustering; regulation of GTPase activity; regulation of protein amino acid phosphorylation; regulation of receptor activity; regulation of synaptic growth at neuromuscular junction; synaptic transmission; synaptogenesis
NCBI Summary:	may play a role in synapse development and regeneration [RGD, Feb 2006]
UniProt Code:	P25304
NCBI GenInfo Identifier:	399021
NCBI Gene ID:	25592
NCBI Accession:	P25304.2
UniProt Secondary Accession:	P25304,Q63034,
UniProt Related Accession:	P25304
Molecular Weight:	208,146 Da
NCBI Full Name:	Agrin
NCBI Synonym Full Names:	agrin
NCBI Official Symbol:	Agrn
NCBI Official Synonym Symbols:	AGR
NCBI Protein Information:	agrin
UniProt Protein Name:	Agrin
Protein Family:	Agrin
UniProt Gene Name:	Agrn
UniProt Entry Name:	AGRIN_RAT

Step	Procedure
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.