Mouse Cell Signalling ELISA Kits 7

Mouse Stromal cell-derived factor 1 (Cxcl12) ELISA Kit

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SKU:
MOEB0393
Product Type:
ELISA Kit
Size:
96 Assays
Uniprot:
P40224
Range:
0.312-20 ng/mL
ELISA Type:
Sandwich
Synonyms:
SDF-1, Stromal Cell Derived Factor 1, SDF1, CXCL12, PBSF, C-X-C motif chemokine 12, Intercrine reduced in hepatomas, IRH, PBSFSDF-1a, Pre-B cell growth-stimulating factor, SCYB12
Reactivity:
Mouse

Description

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Product Name:Mouse Stromal cell-derived factor 1 (Cxcl12) ELISA Kit
Product Code:MOEB0393
Alias:Stromal cell-derived factor 1, SDF-1, 12-O-tetradecanoylphorbol 13-acetate repressed protein 1, TPAR1, C-X-C motif chemokine 12, Pre-B cell growth-stimulating factor, PBSF, Thymic lymphoma cell-stimulating factor, TLSF, Cxcl12, Sdf1
Uniprot:P40224
Reactivity:Mouse
Range:0.312-20 ng/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:CXCL12 iso3: Chemoattractant active on T-lymphocytes, monocytes, but not neutrophils. Activates the C-X-C chemokine receptor CXCR4 to induce a rapid and transient rise in the level of intracellular calcium ions and chemotaxis. Also binds to another C-X-C chemokine receptor CXCR7, which activates the beta-arrestin pathway and acts as a scavenger receptor for SDF-1. SDF-1-beta(3-72) and SDF-1- alpha(3-67) show a reduced chemotactic activity. Binding to cell surface proteoglycans seems to inhibit formation of SDF-1-alpha(3- 67) and thus to preserve activity on local sites. Acts as a positive regulator of monocyte migration and a negative regulator of monocyte adhesion via the LYN kinase. Stimulates migration of monocytes and T-lymphocytes through its receptors, CXCR4 and CXCR7, and decreases monocyte adherence to surfaces coated with ICAM-1, a ligand for beta-2 integrins. SDF1A/CXCR4 signaling axis inhibits beta-2 integrin LFA-1 mediated adhesion of monocytes to ICAM-1 through LYN kinase. Inhibits CXCR4-mediated infection by T- cell line-adapted HIV-1. Plays a protective role after myocardial infarction. Induces down-regulation and internalization of CXCR7 expressed in various cells. Has several critical functions during embryonic development; required for B-cell lymphopoiesis, myelopoiesis in bone marrow and heart ventricular septum formation. Monomer or homodimer; in equilibrium. Dimer formation is induced by non acidic pH and the presence of multivalent anions, and by binding to CXCR4 or heparin. Monomeric form is required for full chemotactic activity and resistance to ischemia/reperfusion injury, whereas the dimeric form acts as a partial agonist of CXCR4, stimulating Ca2+ mobilization but with no chemotactic activity and instead acts as a selective antagonist that blocks chemotaxis induced by the monomeric form. Interacts with the N- terminus of CXCR7. Isoform Alpha and isoform Beta are ubiquitously expressed, with highest levels detected in liver, pancreas and spleen. Isoform Gamma is mainly expressed in heart, with weak expression detected in several other tissues. Isoform Delta, isoform Epsilon and isoform Theta have highest expression levels in pancreas, with lower levels detected in heart, kidney, liver and spleen. Belongs to the intercrine alpha (chemokine CxC) family. 6 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Cell development/differentiation; Secreted, signal peptide; Motility/polarity/chemotaxis; Secreted; Chemokine

Cellular Component: extracellular space; plasma membrane; extracellular region; external side of plasma membrane

Molecular Function:growth factor activity; chemokine receptor binding; chemokine activity; CXCR chemokine receptor binding; cytokine activity

Biological Process: positive regulation of dopamine secretion; organ regeneration; positive regulation of cell adhesion; adult locomotory behavior; neuron migration; motor axon guidance; regulation of calcium ion transport; chemotaxis; regulation of cell migration; germ cell development; induction of positive chemotaxis; T cell proliferation; patterning of blood vessels; ameboidal cell migration; positive regulation of cell proliferation; germ cell migration; positive regulation of axon extension involved in axon guidance; positive regulation of endothelial cell proliferation; immune response; brain development; positive regulation of neuron differentiation; telencephalon cell migration; positive regulation of cell migration

NCBI Summary:This gene encodes a member of the alpha chemokine protein family. The encoded protein is secreted and functions as the ligand for the G-protein coupled receptor, chemokine (C-X-C motif) receptor 4. The encoded protein plays a role in many diverse cellular functions, including embryogenesis, immune surveillance, inflammation response, tissue homeostasis, and tumor growth and metastasis. Alternative splicing results in multiple transcript variants. [provided by RefSeq, May 2013]
UniProt Code:P40224
NCBI GenInfo Identifier:251757334
NCBI Gene ID:20315
NCBI Accession:P40224.2
UniProt Secondary Accession:P40224,Q4FJL5, Q543V6,
UniProt Related Accession:P40224
Molecular Weight:10,032 Da
NCBI Full Name:Stromal cell-derived factor 1
NCBI Synonym Full Names:chemokine (C-X-C motif) ligand 12
NCBI Official Symbol:Cxcl12  
NCBI Official Synonym Symbols:Pbsf; Sdf1; Tlsf; Tpar1; Scyb12  
NCBI Protein Information:stromal cell-derived factor 1; pre-B-cell growth-stimulating factor; thymic lymphoma cell-stimulating factor; 12-O-tetradecanoylphorbol 13-acetate repressed protein 1
UniProt Protein Name:Stromal cell-derived factor 1
UniProt Synonym Protein Names:12-O-tetradecanoylphorbol 13-acetate repressed protein 1; TPAR1; C-X-C motif chemokine 12; Pre-B cell growth-stimulating factor; PBSF; Thymic lymphoma cell-stimulating factor; TLSF
UniProt Gene Name:Cxcl12  
UniProt Entry Name:SDF1_MOUSE
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.
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