Human Forkhead box protein P3 (FOXP3) ELISA Kit

Product Name:	Human Forkhead box protein P3 (FOXP3) ELISA Kit
SKU:	HUEB2005
Size:	96T
Target:	Human Forkhead box protein P3 (FOXP3)
Synonyms:	Scurfin, JM2, IPEX
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.312-20ng/mL
Sensitivity:	0.23ng/mL

Intra CV:

5.7%

Inter CV:

9.5%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	98-108%	102-112%	93-102%	81-91%
EDTA Plasma(N=5)	90-99%	86-95%	85-95%	110-120%
Heparin Plasma(N=5)	85-97%	90-99%	101-111%	80-89%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	101	95-107
Plasma	103	97-109

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 (PubMed:17360565). 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 (PubMed:18354202). Can repress the expression of IL2 and IFNG via its association with transcription factor IKZF4.

Uniprot:	Q9BZS1
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Forkhead box protein P3
Sub Unit:	Homodimer (By similarity). Interacts with IKZF3. Isoform 1 (via LXXLL motif), but not isoform 2, interacts with isoform 4 of RORA (via AF-2 motif). Interacts with STUB1, HSPA8 and HSPA1A/B. Interacts with PPP1CA, PPP1CB and PPP1CG. Interacts with KAT5 and HDAC7. Interacts with HDAC9 in the absence of T-cell stimulation. Interacts with USP7. Interacts with isoform 2 of ZFP90 and can form a complex with TRIM28 in the presence of isoform 2 of ZFP90. Interacts with RUNX1. Interacts with RORC. Interacts with RELA and NFATC2. Interacts with RUNX2, RUNX3 and IKZF4.
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). The 41 kDa form derived by proteolytic processing is found exclusively in the chromatin fraction of activated Treg cells (By similarity).
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; C2H2-type zinc finger protein; DNA-binding; Cell cycle regulation Chromosomal Location of Human Ortholog: Xp11.23 Cellular Component: cytoplasm; nucleus Molecular Function:histone acetyltransferase binding; histone deacetylase binding; NFAT protein binding; protein binding; protein homodimerization activity; sequence-specific DNA binding; transcription factor activity Biological Process: CD4-positive, CD25-positive, alpha-beta regulatory T cell lineage commitment; inhibition of CREB transcription factor; inhibition of NF-kappaB transcription factor; negative regulation of cell proliferation; negative regulation of cytokine biosynthetic process; negative regulation of cytokine secretion; negative regulation of histone deacetylation; negative regulation of immune response; 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 T cell cytokine production; negative regulation of T cell proliferation; negative regulation of transcription factor activity; negative regulation of transcription, DNA-dependent; positive regulation of CD4-positive, CD25-positive, alpha-beta regulatory T cell differentiation; positive regulation of histone acetylation; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; regulation of T cell anergy; response to virus; T cell activation Disease: Diabetes Mellitus, Insulin-dependent; Immunodysregulation, Polyendocrinopathy, And Enteropathy, X-linked
NCBI Summary:	The protein encoded by this gene is a member of the forkhead/winged-helix family of transcriptional regulators. Defects in this gene are the cause of immunodeficiency polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), also known as X-linked autoimmunity-immunodeficiency syndrome. Alternatively spliced transcript variants encoding different isoforms have been identified. [provided by RefSeq, Jul 2008]
UniProt Code:	Q9BZS1
NCBI GenInfo Identifier:	14548061
NCBI Gene ID:	50943
NCBI Accession:	Q9BZS1.1
UniProt Secondary Accession:	Q9BZS1,O60827, Q14DD8, Q4ZH51, A5HJT1, B7ZLG0, B9UN80
UniProt Related Accession:	Q9BZS1
Molecular Weight:	44,407 Da
NCBI Full Name:	Forkhead box protein P3
NCBI Synonym Full Names:	forkhead box P3
NCBI Official Symbol:	FOXP3
NCBI Official Synonym Symbols:	JM2; AIID; IPEX; PIDX; XPID; DIETER
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_HUMAN

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.

ELISA	Antibodies
Human FOXP3 / Forkhead box protein P3 ELISA Kit	Anti-FOXP3 Antibody (CAB12051)
Human FOXP3 (Forkhead Box Protein P3) CLIA Kit (HUES00649)	Anti-FOXP3 Antibody (CAB17147)
Human FOXP3 (Forkhead Box Protein P3) ELISA Kit (HUES02197)	Anti-FOXP3 Antibody (CAB4953)
	Anti-FOXP3 Antibody (CAB5706)
	Anti-FOXP3 Antibody (CAB8024)