Human AKR1C3 ELISA Kit (HUEB0759)- High Sensitivity

Product Name:	Human Aldo-keto reductase family 1 member C3 (AKR1C3) ELISA Kit
Product Code:	HUEB0759
Alias:	Aldo-keto reductase family 1 member C3, 17-beta-hydroxysteroid dehydrogenase type 5, 17-beta-HSD 5, 3-alpha-HSD type II, brain, 3-alpha-hydroxysteroid dehydrogenase type 2, 3-alpha-HSD type 2, Chlordecone reductase homolog HAKRb, Dihydrodiol dehydrogenase 3, DD-3, DD3, Dihydrodiol dehydrogenase type I, HA1753, Indanol dehydrogenase, Prostaglandin F synthase, PGFS, Testosterone 17-beta-dehydrogenase 5
Uniprot:	P42330
Reactivity:	Human
Range:	0.156-10 ng/ml
Detection Method:	Sandwich
Size:	96 Assay
Storage:	Please see kit components below for exact storage details
Note:	For research use only

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

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 AKR1C3 (Aldo-keto reductase family 1 member C3) ELISA Kit (HUFI03431)	Anti-AKR1C3 Antibody (CAB13568)
	Anti-AKR1C3 Antibody (CAB1781)
	Anti-AKR1C3 Antibody (CAB3884)
	RUNX3 Antibody (PACO05593)
	AKR1B1 Antibody (PACO07671)