Human CXCR4 ELISA Kit

SKU:
HUFI01798
€599

Description

ELISA Kit Technical ManualMSDS

Human CXCR4 (C-X-C chemokine receptor type 4) ELISA Kit - Information

The Assay Genie Human CXCR4 (C-X-C chemokine receptor type 4) ELISA Kit can assay for Human CXCR4 in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How our Human CXCR4 (C-X-C chemokine receptor type 4) ELISA Kits Work?

The Assay Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, Assay Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.

At Assay Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound Human CXCR4 is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.

Human CXCR4 (C-X-C chemokine receptor type 4) ELISA Kit Data

Product Code

HUFI01798

UniprotP61073
Alias

CXCR4, Chemokine C-X-C-Motif Receptor, Stromal cell-derived factor 1 receptor, SDF-1 receptor, Leukocyte-derived seven transmembrane domain receptor, LESTR, Fusin, LCR1, CD184, Fusin

Detection method

Sandwich ELISA, Double Antibody

Application

This immunoassay kit allows for the in vitro quantitative determination of Human CXCR4 concentrations in serum plasma and other biological fluids.

Size

96T

Range

78.125-5000pg/ml

Sensitivity

46.875pg/ml

Storage

4'C for 6 months

Recovery

Matrices listed below were spiked with certain level of Human CXCR4 and the recovery rates were calculated by comparing the measured value to the expected amount of Human CXCR4 in samples.

MatrixRecovery range(%)Average(%)
serum(n=5)89-10395
EDTA plasma(n=5)86-9990
UFH plasma(n=5)85-10592
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human CXCR4 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample1:21:41:8
serum(n=5)88-103%88-105%91-104%
EDTA plasma(n=5)93-97%83-95%84-96%
UFH plasma(n=5)82-99%93-99%80-96%
CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Note

For Research Use Only

Human CXCR4 (C-X-C chemokine receptor type 4) ELISA Kit Protocol

The below protocol is a sample protocol for Human CXCR4 (C-X-C chemokine receptor type 4) ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISA Kits allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Human CXCR4 Antibody present in their sample.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Assay Protocol:

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.

Human CXCR4 (C-X-C chemokine receptor type 4) ELISA Kit components

96 Assays

Storage

ELISA Microplate (Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5 -

Other materials and equipment required:

The Assay Genie Human CXCR4 (C-X-C chemokine receptor type 4) ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
  • 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

Sample Preparation

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 TypeProtocol

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.

Human CXCR4 Protein Information

UniProt Protein Function:CXCR4: Receptor for the C-X-C chemokine CXCL12/SDF-1 that transduces a signal by increasing intracellular calcium ion levels and enhancing MAPK1/MAPK3 activation. Acts as a receptor for extracellular ubiquitin; leading to enhanced intracellular calcium ions and reduced cellular cAMP levels. Involved in hematopoiesis and in cardiac ventricular septum formation. Also plays an essential role in vascularization of the gastrointestinal tract, probably by regulating vascular branching and/or remodeling processes in endothelial cells. Involved in cerebellar development. In the CNS, could mediate hippocampal-neuron survival. Acts as a coreceptor (CD4 being the primary receptor) for HIV-1 X4 isolates and as a primary receptor for some HIV-2 isolates. Promotes Env-mediated fusion of the virus. Monomer. Can form dimers. Interacts with CD164. Interacts with HIV-1 surface protein gp120 and Tat. Interacts with ARRB2; the interaction is dependent on the C-terminal phosphorylation of CXCR4 and allows activation of MAPK1 and MAPK3. Interacts with ARRC; the interaction is dependent on the C-terminal phosphorylation of CXCR4 and modulates calcium mobilization. Interacts (via the cytoplasmic C-terminal) with ITCH (via the WW domains I and II); the interaction, enhanced by CXCL12, ubiquitinates CXCR4 and leads to its degradation. Interacts with extracellular ubiquitin. Interacts with human cytomegalovirus/HHV- 5 protein UL78. Expressed in numerous tissues, such as peripheral blood leukocytes, spleen, thymus, spinal cord, heart, placenta, lung, liver, skeletal muscle, kidney, pancreas, cerebellum, cerebral cortex and medulla (in microglia as well as in astrocytes), brain microvascular, coronary artery and umbilical cord endothelial cells. Isoform 1 is predominant in all tissues tested. Belongs to the G-protein coupled receptor 1 family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Membrane protein, integral; Receptor, GPCR; GPCR, family 1; Membrane protein, multi-pass

Chromosomal Location of Human Ortholog: 2q21

Cellular Component: cell surface; lysosome; leading edge; early endosome; cytoplasmic membrane-bound vesicle; integral to membrane; intercellular junction; growth cone; late endosome; cytoplasm; plasma membrane; cytoplasmic vesicle; external side of plasma membrane

Molecular Function:G-protein coupled receptor activity; viral receptor activity; ubiquitin binding; protein binding; cytokine binding; ubiquitin protein ligase binding; myosin light chain binding; coreceptor activity; actin binding; C-X-C chemokine receptor activity

Biological Process: entry of virus into host cell; regulation of chemotaxis; viral reproduction; activation of MAPK activity; apoptosis; neuron migration; motor axon guidance; regulation of cell migration; germ cell development; T cell proliferation; elevation of cytosolic calcium ion concentration; germ cell migration; dendritic cell chemotaxis; positive regulation of oligodendrocyte differentiation; inflammatory response; neutrophil activation; response to virus; calcium-mediated signaling; patterning of blood vessels; G-protein coupled receptor protein signaling pathway; ameboidal cell migration; myelin maintenance; response to hypoxia; entry into host cell; brain development

Disease: Whim Syndrome

NCBI Summary:This gene encodes a CXC chemokine receptor specific for stromal cell-derived factor-1. The protein has 7 transmembrane regions and is located on the cell surface. It acts with the CD4 protein to support HIV entry into cells and is also highly expressed in breast cancer cells. Mutations in this gene have been associated with WHIM (warts, hypogammaglobulinemia, infections, and myelokathexis) syndrome. Alternate transcriptional splice variants, encoding different isoforms, have been characterized. [provided by RefSeq, Jul 2008]
UniProt Code:P61073
NCBI GenInfo Identifier:46577576
NCBI Gene ID:7852
NCBI Accession:P61073.1
UniProt Secondary Accession:P61073,O60835, P30991, P56438, Q53S69, Q9BXA0, Q9UKN2 B2R5N0,
UniProt Related Accession:P61073
Molecular Weight:40,221 Da
NCBI Full Name:C-X-C chemokine receptor type 4
NCBI Synonym Full Names:chemokine (C-X-C motif) receptor 4
NCBI Official Symbol:CXCR4
NCBI Official Synonym Symbols:FB22; HM89; LAP3; LCR1; NPYR; WHIM; CD184; LAP-3; LESTR; NPY3R; NPYRL; HSY3RR; NPYY3R; D2S201E
NCBI Protein Information:C-X-C chemokine receptor type 4; fusin; CD184 antigen; SDF-1 receptor; LPS-associated protein 3; neuropeptide Y receptor Y3; seven transmembrane helix receptor; stromal cell-derived factor 1 receptor; lipopolysaccharide-associated protein 3; seven-transmembrane-segment receptor, spleen; leukocyte-derived seven transmembrane domain receptor
UniProt Protein Name:C-X-C chemokine receptor type 4
UniProt Synonym Protein Names:FB22; Fusin; HM89; LCR1; Leukocyte-derived seven transmembrane domain receptor; LESTR; Lipopolysaccharide-associated protein 3; LAP-3; LPS-associated protein 3; NPYRL; Stromal cell-derived factor 1 receptor; SDF-1 receptor; CD_antigen: CD184
Protein Family:C-X-C chemokine receptor
UniProt Gene Name:CXCR4
UniProt Entry Name:CXCR4_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
Research Area:
Immunology
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