Mouse Forkhead box protein P3 (Foxp3) ELISA Kit

Product Name:	Mouse Forkhead box protein P3 (Foxp3) ELISA Kit
SKU:	MOEB1746
Size:	96T
Target:	Mouse Forkhead box protein P3 (Foxp3)
Synonyms:	Scurfin
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Mouse
Detection Range:	0.25-16ng/mL
Sensitivity:	0.125ng/mL

Intra CV:

6.4%

Inter CV:

8.2%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	98-107%	103-112%	109-118%	94-104%
EDTA Plasma(N=5)	105-114%	99-109%	87-99%	105-115%
Heparin Plasma(N=5)	104-113%	88-97%	107-119%	106-116%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	100	94-106
Plasma	102	96-108

Function:

Transcriptional regulator which is crucial for the development and inhibitory function of regulatory T-cells (Treg). Plays an essential role in maintaining homeostasis of the immune system by allowing the acquisition of full suppressive function and stability of the Treg lineage, and by directly modulating the expansion and function of conventional T-cells. Can act either as a transcriptional repressor or a transcriptional activator depending on its interactions with other transcription factors, histone acetylases and deacetylases. The suppressive activity of Treg involves the coordinate activation of many genes, including CTLA4 and TNFRSF18 by FOXP3 along with repression of genes encoding cytokines such as interleukin-2 (IL2) and interferon-gamma (IFNG). Inhibits cytokine production and T-cell effector function by repressing the activity of two key transcription factors, RELA and NFATC2 (PubMed:15790681). Mediates transcriptional repression of IL2 via its association with histone acetylase KAT5 and histone deacetylase HDAC7 (By similarity). Can activate the expression of TNFRSF18, IL2RA and CTLA4 and repress the expression of IL2 and IFNG via its association with transcription factor RUNX1 (PubMed:17377532). Inhibits the differentiation of IL17 producing helper T-cells (Th17) by antagonizing RORC function, leading to down-regulation of IL17 expression, favoring Treg development (PubMed:18368049). Inhibits the transcriptional activator activity of RORA (By similarity). Can repress the expression of IL2 and IFNG via its association with transcription factor IKZF4 (PubMed:19696312).

Uniprot:	Q99JB6
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant mouse Forkhead box protein P3
Sub Unit:	Homodimer. Interacts with IKZF3 (By similarity). Interacts (via LXXLL motif) with isoform 4 of RORA (via AF-2 motif) (By similarity). Interacts with STUB1 and HSPA1A/B. Interacts with IKZF4, HDAC7 and KAT5. Interacts with RUNX1, RUNX2, RUNX3 and NFATC2. Interacts with RORC. Interacts with HDAC9 in the absence of T-cell stimulation (By similarity). Interacts with RELA, PPP1CA, PPP1CB, PPP1CG, HSPA8 and USP7.
Research Area:	Cancer
Subcellular Location:	Nucleus Cytoplasm Predominantly expressed in the cytoplasm in activated conventional T-cells whereas predominantly expressed in the nucleus in regulatory T-cells (Treg) (By similarity). The 41 kDa form derived by proteolytic processing is found exclusively in the chromatin fraction of activated Treg cells.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	FOXP3: Probable transcription factor. Plays a critical role in the control of immune response. Interacts with IKZF3. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Transcription factor; Cell cycle regulation; DNA-binding; C2H2-type zinc finger protein Cellular Component: cytoplasm; intracellular; nucleus Molecular Function:DNA binding; histone acetyltransferase binding; histone deacetylase binding; metal ion binding; NFAT protein binding; nucleic acid binding; protein binding; protein homodimerization activity; RNA polymerase II transcription factor activity, enhancer binding; sequence-specific DNA binding; transcription corepressor activity; transcription factor activity Biological Process: B cell homeostasis; CD4-positive, CD25-positive, alpha-beta regulatory T cell differentiation; CD4-positive, CD25-positive, alpha-beta regulatory T cell lineage commitment; cytokine production; inhibition of CREB transcription factor; inhibition of NF-kappaB transcription factor; myeloid cell homeostasis; negative regulation of cell proliferation; negative regulation of chronic inflammatory response; negative regulation of cytokine biosynthetic process; negative regulation of cytokine secretion; negative regulation of histone acetylation; negative regulation of histone deacetylation; negative regulation of immune response; negative regulation of inflammatory response; negative regulation of interferon-gamma biosynthetic process; negative regulation of interferon-gamma production; negative regulation of interleukin-10 production; negative regulation of interleukin-17 production; negative regulation of interleukin-2 biosynthetic process; negative regulation of interleukin-2 production; negative regulation of interleukin-4 production; negative regulation of interleukin-5 production; negative regulation of interleukin-6 production; negative regulation of isotype switching to IgE isotypes; negative regulation of lymphocyte proliferation; negative regulation of T cell cytokine production; negative regulation of T cell proliferation; negative regulation of transcription factor activity; negative regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; negative regulation of tumor necrosis factor production; positive regulation of CD4-positive, CD25-positive, alpha-beta regulatory T cell differentiation; positive regulation of histone acetylation; positive regulation of immature T cell proliferation in the thymus; positive regulation of interleukin-4 production; positive regulation of peripheral T cell tolerance induction; positive regulation of regulatory T cell differentiation; positive regulation of T cell anergy; positive regulation of T cell tolerance induction; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; positive regulation of transforming growth factor-beta1 production; regulation of immunoglobulin production; regulation of isotype switching to IgG isotypes; regulation of T cell anergy; regulation of transcription from RNA polymerase II promoter; regulation of transcription, DNA-dependent; regulatory T cell differentiation; T cell activation; T cell mediated immunity; T cell receptor signaling pathway; tolerance induction; tolerance induction to self antigen; transcription, DNA-dependent
NCBI Summary:	The protein encoded by this gene is a member of the forkhead/winged-helix family of transcriptional regulators. Defects in this gene result in the scurfy phenotype (sf). Alternative splicing results in multiple transcript variants. [provided by RefSeq, Sep 2015]
UniProt Code:	Q99JB6
NCBI GenInfo Identifier:	16905075
NCBI Gene ID:	20371
NCBI Accession:	NP_473380.1
UniProt Related Accession:	Q99JB6
Molecular Weight:
NCBI Full Name:	forkhead box protein P3
NCBI Synonym Full Names:	forkhead box P3
NCBI Official Symbol:	Foxp3
NCBI Official Synonym Symbols:	sf; JM2; scurfin
NCBI Protein Information:	forkhead box protein P3
UniProt Protein Name:	Forkhead box protein P3
UniProt Synonym Protein Names:	Scurfin
Protein Family:	Forkhead box protein
UniProt Gene Name:	Foxp3
UniProt Entry Name:	FOXP3_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.