Rat Semaphorin 3A / SEMA3A ELISA Kit

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SKU:
RTFI01121
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
Q63548
Sensitivity:
0.469ng/ml
Range:
0.781-50ng/ml
ELISA Type:
Sandwich ELISA, Double Antibody
Synonyms:
SEMA3A, coll-1, collapsin 1, Hsema-I, Hsema-III, sema domain, immunoglobulin domain, Ig, short basic domain, secreted, Sema III, SEMA1, SEMAD, SEMAIII, SEMAL, semaphorin D, Semaphorin III, semaphorin L, semaphorin-3A, SemD, Semaphorin III, Sema III
Reactivity:
Rat
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Description

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Product Name:Rat SEMA3A (Semaphorin-3A) ELISA Kit
Product Code:RTFI01121
Size:96 Assays
Target:Rat SEMA3A
Alias:SEMA3A, coll-1, collapsin 1, Hsema-I, Hsema-III, sema domain, immunoglobulin domain, Ig, short basic domain, secreted, Sema III, SEMA1, SEMAD, SEMAIII, SEMAL, semaphorin D, Semaphorin III, semaphorin L, semaphorin-3A, SemD, Semaphorin III, Sema III
Reactivity:Rat
Detection Method:Sandwich ELISA, Double Antibody
Sensitivity:0.469ng/ml
Range:0.781-50ng/ml
Storage:4°C for 6 months
Note:For Research Use Only
Recovery:Matrices listed below were spiked with certain level of Rat SEMA3A and the recovery rates were calculated by comparing the measured value to the expected amount of Rat SEMA3A in samples.
MatrixRecovery range(%)Average(%)
serum(n=5)88-10597
EDTA plasma(n=5)88-10597
UFH plasma(n=5)89-10395
Linearity:The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat SEMA3A and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
Sample1:21:41:8
serum(n=5)85-95%91-99%86-103%
EDTA plasma(n=5)89-99%83-94%82-95%
UFH plasma(n=5)88-99%84-96%89-99%
Intra-Assay:CV <8%
Inter-Assay:CV <10%
Uniprot:Q63548
UniProt Protein Function:SEMA3A: May be involved in guiding growing axons towards their targets by forming a molecular boundary that instructs axons to engage in the formation of specific nerve tracts. Binds to neuropilin. Involved in the development of the olfactory system and in neuronal control of puberty (By similarity)
UniProt Protein Details:

Protein type:Secreted, signal peptide; Secreted; Motility/polarity/chemotaxis

Cellular Component: axon; dendrite; extracellular space

Molecular Function:chemorepellent activity; semaphorin receptor binding

Biological Process: apoptosis; axon extension involved in axon guidance; axon guidance; axonal fasciculation; branchiomotor neuron axon guidance; dendrite morphogenesis; facial nerve structural organization; gonadotrophin-releasing hormone neuronal migration to the hypothalamus; motor axon guidance; negative chemotaxis; negative regulation of axon extension; negative regulation of axon extension involved in axon guidance; nerve development; neural crest cell migration; neuron migration; olfactory bulb development; regulation of axon extension involved in axon guidance; regulation of heart rate; trigeminal nerve structural organization

NCBI Summary:induces neuronal cell death; induces apoptosis of retinal ganglion cells in response to injury [RGD, Feb 2006]
UniProt Code:Q63548
NCBI GenInfo Identifier:8394255
NCBI Gene ID:29751
NCBI Accession:NP_059006.1
UniProt Related Accession:Q63548
Molecular Weight:90.6 kDa
NCBI Full Name:semaphorin-3A
NCBI Synonym Full Names:semaphorin 3A
NCBI Official Symbol:Sema3a  
NCBI Protein Information:semaphorin-3A
UniProt Protein Name:Semaphorin-3A
UniProt Synonym Protein Names:Semaphorin III; Sema III
Protein Family:Semaphorin
UniProt Gene Name:Sema3a  
UniProt Entry Name:SEM3A_RAT
StepProcedure
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.
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