Human ABCG2 ELISA Kit (HUEB0916)- High Sensitivity

Product Name:	Human ATP-binding cassette sub-family G member 2 (ABCG2) ELISA Kit
SKU:	HUEB0916
Size:	96T
Target:	Human ATP-binding cassette sub-family G member 2 (ABCG2)
Synonyms:	Breast cancer resistance protein, CDw338, Mitoxantrone resistance-associated protein, Placenta-specific ATP-binding cassette transporter, Urate exporter, CD338, ABCP, BCRP, BCRP1, MXR
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.156-10ng/mL
Sensitivity:	0.067ng/mL

Intra CV:

3.8%

Inter CV:

7.2%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	80-89%	80-90%	107-117%	105-115%
EDTA Plasma(N=5)	108-117%	89-99%	102-114%	99-108%
Heparin Plasma(N=5)	91-101%	98-108%	82-91%	85-97%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	107	101-113
Plasma	109	103-115

Function:

High-capacity urate exporter functioning in both renal and extrarenal urate excretion. Plays a role in porphyrin homeostasis as it is able to mediates the export of protoporhyrin IX (PPIX) both from mitochondria to cytosol and from cytosol to extracellular space, and cellular export of hemin, and heme. Xenobiotic transporter that may play an important role in the exclusion of xenobiotics from the brain. Appears to play a major role in the multidrug resistance phenotype of several cancer cell lines. Implicated in the efflux of numerous drugs and xenobiotics: mitoxantrone, the photosensitizer pheophorbide, camptothecin, methotrexate, azidothymidine (AZT), and the anthracyclines daunorubicin and doxorubicin.

Uniprot:	Q9UNQ0
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human ATP-binding cassette sub-family G member 2
Sub Unit:	Monomer under reducing conditions, the minimal functional units is a homodimer; disulfide-linked, but the major oligomeric form in plasma membranes is a homotetramer with possibility of higher order oligomerization up to homododecamers.
Research Area:	Neurosciences
Subcellular Location:	Cell membrane Multi-pass membrane protein Mitochondrion membrane Multi-pass membrane protein
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	ABCG2: Xenobiotic transporter that may play an important role in the exclusion of xenobiotics from the brain. May be involved in brain-to-blood efflux. Appears to play a major role in the multidrug resistance phenotype of several cancer cell lines. When overexpressed, the transfected cells become resistant to mitoxantrone, daunorubicin and doxorubicin, display diminished intracellular accumulation of daunorubicin, and manifest an ATP- dependent increase in the efflux of rhodamine 123. Monomer or homodimer; disulfide-linked. Up-regulated in brain tumors. Highly expressed in placenta. Low expression in small intestine, liver and colon. Belongs to the ABC transporter superfamily. ABCG family. Eye pigment precursor importer (TC 3.A.1.204) subfamily. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Membrane protein, integral; Transporter; Transporter, ABC family; Membrane protein, multi-pass; Transporter, iron Chromosomal Location of Human Ortholog: 4q22 Cellular Component: nucleoplasm; mitochondrial membrane; plasma membrane; integral to membrane; nucleus Molecular Function:protein binding; protein homodimerization activity; transporter activity; ATPase activity, coupled to transmembrane movement of substances; heme transporter activity; ATP binding; xenobiotic-transporting ATPase activity Biological Process: response to drug; heme transport; urate metabolic process; transport; cellular iron ion homeostasis; xenobiotic transport; multidrug transport; transmembrane transport Disease: Blood Group, Junior System; Uric Acid Concentration, Serum, Quantitative Trait Locus 1
NCBI Summary:	The membrane-associated protein encoded by this gene is included in the superfamily of ATP-binding cassette (ABC) transporters. ABC proteins transport various molecules across extra- and intra-cellular membranes. ABC genes are divided into seven distinct subfamilies (ABC1, MDR/TAP, MRP, ALD, OABP, GCN20, White). This protein is a member of the White subfamily. Alternatively referred to as a breast cancer resistance protein, this protein functions as a xenobiotic transporter which may play a major role in multi-drug resistance. It likely serves as a cellular defense mechanism in response to mitoxantrone and anthracycline exposure. Significant expression of this protein has been observed in the placenta, which may suggest a potential role for this molecule in placenta tissue. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Apr 2012]
UniProt Code:	Q9UNQ0
NCBI GenInfo Identifier:	67462103
NCBI Gene ID:	9429
NCBI Accession:	Q9UNQ0.3
UniProt Secondary Accession:	Q9UNQ0,O95374, Q4W5I3, Q53ZQ1, Q569L4, Q5YLG4, Q86V64 Q8IX16, Q96LD6, Q96TA8, A0A1W3, A8K1T5,
UniProt Related Accession:	Q9UNQ0
Molecular Weight:	72,314 Da
NCBI Full Name:	ATP-binding cassette sub-family G member 2
NCBI Synonym Full Names:	ATP-binding cassette, sub-family G (WHITE), member 2
NCBI Official Symbol:	ABCG2
NCBI Official Synonym Symbols:	MRX; MXR; ABCP; BCRP; BMDP; MXR1; ABC15; BCRP1; CD338; GOUT1; CDw338; UAQTL1; EST157481
NCBI Protein Information:	ATP-binding cassette sub-family G member 2; urate exporter; ABC transporter; placenta specific MDR protein; breast cancer resistance protein; ATP-binding cassette transporter G2; mitoxantrone resistance-associated protein; placenta-specific ATP-binding cassette transporter; multi drug resistance efflux transport ATP-binding cassette sub-family G (WHITE) member 2
UniProt Protein Name:	ATP-binding cassette sub-family G member 2
UniProt Synonym Protein Names:	Breast cancer resistance protein; CDw338; Mitoxantrone resistance-associated protein; Placenta-specific ATP-binding cassette transporter; Urate exporter
UniProt Gene Name:	ABCG2
UniProt Entry Name:	ABCG2_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 BCRP / ABCG2 ELISA Kit	Anti-ABCG2 Antibody (CAB17908)
Human ABCG2 (ATP Binding Cassette Transporter G2) CLIA Kit (HUES00372)	Anti-ABCG2 Antibody (CAB5661)
Human ABCG2 (ATP Binding Cassette Transporter G2) ELISA Kit (HUES01726)	ABCG2 Antibody (PACO02223)
	ABCF2 Antibody (PACO06218)
	CTNNB1 Antibody (PACO13380)