Product Name:	Human UGT1A1 / UDP-glucuronosyltransferase 1-1 ELISA Kit
Product Code:	HUFI01190
Size:	96 Assays
Alias:	UGT1A1, UDP-glucuronosyltransferase 1-1, UDPGT 1-1, bilirubin UDP-glucuronosyltransferase 1-1, UDP glucuronosyltransferase 1 family, polypeptide A1, UGT1*1, UGT1-01, UGT1.1, UDP-glucuronosyltransferase 1A1, Bilirubin-specific UDPGT isozyme 1, hUG-BR1, UDP-glucuronosyltransferase 1-A, UGT-1A, UGT1A, GNT1, UGT1
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human UGT1A1 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.094ng/ml
Range:	0.156-10ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human UGT1A1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human UGT1A1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-102	96
EDTA plasma(n=5)	93-100	97
UFH plasma(n=5)	85-96	92

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human UGT1A1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	85-101%	85-95%	88-97%
EDTA plasma(n=5)	82-101%	83-98%	86-98%
UFH plasma(n=5)	84-98%	81-93%	83-99%

CV(%):

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

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	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

Uniprot

P22309

UniProt Protein Function:	UGT1A1: UDPGT is of major importance in the conjugation and subsequent elimination of potentially toxic xenobiotics and endogenous compounds. This isoform glucuronidates bilirubin IX- alpha to form both the IX-alpha-C8 and IX-alpha-C12 monoconjugates and diconjugate. Is also able to catalyze the glucuronidation of 17beta-estradiol, 17alpha-ethinylestradiol, 1-hydroxypyrene, 4- methylumbelliferone, 1-naphthol, paranitrophenol, scopoletin, and umbelliferone. Part a large chaperone multiprotein complex comprising DNAJB11, HSP90B1, HSPA5, HYOU, PDIA2, PDIA4, PDIA6, PPIB, SDF2L1, UGT1A1 and very small amounts of ERP29, but not, or at very low levels, CALR nor CANX. Expressed in liver. Not expressed in skin or kidney. Belongs to the UDP-glycosyltransferase family. 1 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Lipid Metabolism - androgen and estrogen; Xenobiotic Metabolism - drug metabolism - other enzymes; Xenobiotic Metabolism - metabolism by cytochrome P450; Xenobiotic Metabolism - drug metabolism - cytochrome P450; Cofactor and Vitamin Metabolism - porphyrin and chlorophyll; Membrane protein, integral; Cofactor and Vitamin Metabolism - retinol; Carbohydrate Metabolism - starch and sucrose; Carbohydrate Metabolism - pentose and glucuronate interconversions; Carbohydrate Metabolism - ascorbate and aldarate; EC 2.4.1.17; Transferase Chromosomal Location of Human Ortholog: 2q37 Cellular Component: endoplasmic reticulum membrane; integral to plasma membrane; endoplasmic reticulum Molecular Function:enzyme inhibitor activity; protein homodimerization activity; retinoic acid binding; enzyme binding; glucuronosyltransferase activity; protein heterodimerization activity; steroid binding Biological Process: response to drug; steroid metabolic process; estrogen metabolic process; negative regulation of steroid metabolic process; organ regeneration; response to lipopolysaccharide; negative regulation of fatty acid metabolic process; liver development; cellular response to hormone stimulus; response to starvation; negative regulation of transferase activity; bilirubin conjugation; flavonoid biosynthetic process; heme catabolic process; xenobiotic metabolic process; negative regulation of catalytic activity; porphyrin metabolic process; digestion; flavone metabolic process; acute-phase response; retinoic acid metabolic process; heterocycle metabolic process; drug metabolic process; response to nutrient Disease: Gilbert Syndrome; Crigler-najjar Syndrome, Type I; Bilirubin, Serum Level Of, Quantitative Trait Locus 1; Crigler-najjar Syndrome, Type Ii; Hyperbilirubinemia, Transient Familial Neonatal
NCBI Summary:	This gene encodes a UDP-glucuronosyltransferase, an enzyme of the glucuronidation pathway that transforms small lipophilic molecules, such as steroids, bilirubin, hormones, and drugs, into water-soluble, excretable metabolites. This gene is part of a complex locus that encodes several UDP-glucuronosyltransferases. The locus includes thirteen unique alternate first exons followed by four common exons. Four of the alternate first exons are considered pseudogenes. Each of the remaining nine 5' exons may be spliced to the four common exons, resulting in nine proteins with different N-termini and identical C-termini. Each first exon encodes the substrate binding site, and is regulated by its own promoter. The preferred substrate of this enzyme is bilirubin, although it also has moderate activity with simple phenols, flavones, and C18 steroids. Mutations in this gene result in Crigler-Najjar syndromes types I and II and in Gilbert syndrome. [provided by RefSeq, Jul 2008]
UniProt Code:	P22309
NCBI GenInfo Identifier:	136729
NCBI Gene ID:	54658
NCBI Accession:	P22309.1
UniProt Secondary Accession:	P22309,A6NJC3, B8K286,
UniProt Related Accession:	P22309
Molecular Weight:	533
NCBI Full Name:	UDP-glucuronosyltransferase 1-1
NCBI Synonym Full Names:	UDP glucuronosyltransferase 1 family, polypeptide A1
NCBI Official Symbol:	UGT1A1
NCBI Official Synonym Symbols:	GNT1; UGT1; UDPGT; UGT1A; HUG-BR1; BILIQTL1; UDPGT 1-1
NCBI Protein Information:	UDP-glucuronosyltransferase 1-1; UGT-1A; UGT1*1; UGT1-01; UDP-glucuronosyltransferase 1-A; UDP-glucuronosyltransferase 1A1; bilirubin-specific UDPGT isozyme 1; bilirubin UDP-glucuronosyltransferase 1-1; bilirubin UDP-glucuronosyltransferase isozyme 1; UDP glycosyltransferase 1 family, polypeptide A1
UniProt Protein Name:	UDP-glucuronosyltransferase 1-1
UniProt Synonym Protein Names:	Bilirubin-specific UDPGT isozyme 1; hUG-BR1; UDP-glucuronosyltransferase 1-A; UGT-1A; UGT1A; UDP-glucuronosyltransferase 1A1
Protein Family:	UDP-glucuronosyltransferase
UniProt Gene Name:	UGT1A1
UniProt Entry Name:	UD11_HUMAN

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

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.

Step	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.

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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Human UGT1A1 / UDP-glucuronosyltransferase 1-1 ELISA Kit

Description