Product Name:	Human AKR1C3 (Aldo-keto reductase family 1 member C3) ELISA Kit
Product Code:	HUFI03431
Size:	96 Assays
Alias:	Aldo-keto reductase family 1 member C3 ELISA Kit, AKR1C3 ELISA Kit, 17-beta-hydroxysteroid dehydrogenase type 5 ELISA Kit, 17-beta-HSD 5 ELISA Kit, 3-alpha-HSD type II ELISA Kit, brain ELISA Kit, 3-alpha-hydroxysteroid dehydrogenase type 2 ELISA Kit, 3-alpha-HSD type 2 ELISA Kit, Chlordecone reductase homolog HAKRb ELISA Kit, Dihydrodiol dehydrogenase 3 ELISA Kit, DD-3 ELISA Kit, DD3 ELISA Kit, Dihydrodiol dehydrogenase type I ELISA Kit, HA1753 ELISA Kit, Indanol dehydrogenase ELISA Kit, Prostaglandin F synthase ELISA Kit, PGFS ELISA Kit, Testosterone 17-beta-dehydrogenase 5 ELISA Kit, Trans-1 ELISA Kit, 2-dihydrobenzene-1 ELISA Kit, 2-diol dehydrogenase ELISA Kit, HSD17B5 ELISA Kit, KIAA0119 ELISA Kit, DDH1 ELISA Kit
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human AKR1C3 (Aldo-keto reductase family 1 member C3) 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 AKR1C3 (Aldo-keto reductase family 1 member C3) and the recovery rates were calculated by comparing the measured value to the expected amount of Human AKR1C3 (Aldo-keto reductase family 1 member C3) in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-101	96
EDTA plasma(n=5)	91-103	97
heparin plasma(n=5)	85-105	93

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human AKR1C3 (Aldo-keto reductase family 1 member C3) 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)	85-103%	86-103%	88-97%
EDTA plasma(n=5)	86-99%	83-95%	85-94%
UFH plasma(n=5)	84-99%	82-100%	84-96%

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 Protein Function:	AKR1C3: Catalyzes the conversion of aldehydes and ketones to alcohols. Catalyzes the reduction of prostaglandin (PG) D2, PGH2 and phenanthrenequinone (PQ) and the oxidation of 9-alpha,11-beta- PGF2 to PGD2. Functions as a bi-directional 3-alpha-, 17-beta- and 20-alpha HSD. Can interconvert active androgens, estrogens and progestins with their cognate inactive metabolites. Preferentially transforms androstenedione (4-dione) to testosterone. Belongs to the aldo/keto reductase family.
UniProt Protein Details:	Protein type:Lipid Metabolism - arachidonic acid; Oxidoreductase; EC 1.1.1.149; Xenobiotic Metabolism - metabolism by cytochrome P450 Chromosomal Location of Human Ortholog: 10p15-p14 Cellular Component: cytoplasm; intracellular; nucleus; cytosol Molecular Function:15-hydroxyprostaglandin-D dehydrogenase (NADP+) activity; delta4-3-oxosteroid 5beta-reductase activity; aldo-keto reductase activity; prostaglandin-F synthase activity; 3-alpha(17-beta)-hydroxysteroid dehydrogenase (NAD+) activity; 3(or 17)-alpha-hydroxysteroid dehydrogenase activity; retinol dehydrogenase activity; phenanthrene 9,10-monooxygenase activity; oxidoreductase activity, acting on NADH or NADPH, quinone or similar compound as acceptor; testosterone 17-beta-dehydrogenase (NADP+) activity; ketosteroid monooxygenase activity; trans-1,2-dihydrobenzene-1,2-diol dehydrogenase activity; ketoreductase activity; indanol dehydrogenase activity; aldehyde reductase activity; geranylgeranyl reductase activity; retinal dehydrogenase activity Biological Process: steroid metabolic process; phototransduction, visible light; retinal metabolic process; cyclooxygenase pathway; male gonad development; progesterone metabolic process; cellular response to starvation; prostaglandin metabolic process; keratinocyte differentiation; G-protein coupled receptor protein signaling pathway; positive regulation of protein kinase B signaling cascade; protein import into nucleus, translocation; positive regulation of cell proliferation; arachidonic acid metabolic process; multicellular organismal macromolecule metabolic process; farnesol catabolic process; retinoid metabolic process; regulation of retinoic acid receptor signaling pathway; response to nutrient
NCBI Summary:	This gene encodes a member of the aldo/keto reductase superfamily, which consists of more than 40 known enzymes and proteins. These enzymes catalyze the conversion of aldehydes and ketones to their corresponding alcohols by utilizing NADH and/or NADPH as cofactors. The enzymes display overlapping but distinct substrate specificity. This enzyme catalyzes the reduction of prostaglandin (PG) D2, PGH2 and phenanthrenequinone (PQ), and the oxidation of 9alpha,11beta-PGF2 to PGD2. It may play an important role in the pathogenesis of allergic diseases such as asthma, and may also have a role in controlling cell growth and/or differentiation. This gene shares high sequence identity with three other gene members and is clustered with those three genes at chromosome 10p15-p14. Three transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Dec 2011]
UniProt Code:	P42330
NCBI GenInfo Identifier:	308153646
NCBI Gene ID:	8644
NCBI Accession:	P42330.4
UniProt Related Accession:	P42330
Molecular Weight:
NCBI Full Name:	Aldo-keto reductase family 1 member C3
NCBI Synonym Full Names:	aldo-keto reductase family 1 member C3
NCBI Official Symbol:	AKR1C3
NCBI Official Synonym Symbols:	DD3; DDX; PGFS; HAKRB; HAKRe; HA1753; HSD17B5; hluPGFS
NCBI Protein Information:	aldo-keto reductase family 1 member C3
UniProt Protein Name:	Aldo-keto reductase family 1 member C3
UniProt Synonym Protein Names:	17-beta-hydroxysteroid dehydrogenase type 5; 17-beta-HSD 5; 3-alpha-HSD type II, brain; 3-alpha-hydroxysteroid dehydrogenase type 2 (EC:1.1.1.357); 3-alpha-HSD type 2; Chlordecone reductase homolog HAKRb; Dihydrodiol dehydrogenase 3; DD-3; DD3; Dihydrodiol dehydrogenase type I; HA1753; Indanol dehydrogenase (EC:1.1.1.112); Prostaglandin F synthase (EC:1.1.1.188); PGFS; Testosterone 17-beta-dehydrogenase 5 (EC:1.1.1.239, EC:1.1.1.64); Trans-1,2-dihydrobenzene-1,2-diol dehydrogenase (EC:1.3.1.20)
UniProt Gene Name:	AKR1C3
UniProt Entry Name:	AK1C3_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.

Antibodies

Anti-AKR1C3 Antibody (CAB13568)

Anti-AKR1C3 Antibody (CAB1781)

Anti-AKR1C3 Antibody (CAB3884)

AKR1C3 Antibody (PACO05594)

AKR1C3 Antibody (PACO07672)