Akr1c3

Manufactured by Merck Group

Sourced in United States

AKR1C3 is a laboratory enzyme that catalyzes the interconversion of carbonyl compounds and alcohols. It is commonly used in various research applications that require the manipulation of these types of chemical species.

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Lab products found in correlation

Enhanced chemiluminescence detection system, by Merck Group (2 mentions) Hsd3b, by Santa Cruz Biotechnology (2 mentions) Ripa buffer, by Thermo Fisher Scientific (2 mentions) Ar n 20 sc 816, by Santa Cruz Biotechnology (1 mentions) Vectastain abc kit, by Vector Laboratories (1 mentions) Antigen retrieval citra plus solution, by BioGenex (1 mentions) 3 3 diaminobenzidine tetrahydrochloride, by Vector Laboratories (1 mentions) Blocking serum from abc vectastain kit, by Vector Laboratories (1 mentions) Mayer s hematoxylin, by Merck Group (1 mentions) Permount, by Merck Group (1 mentions) Np6 g6 a6, by Merck Group (1 mentions) Cyp17a1, by Santa Cruz Biotechnology (1 mentions) Blocking buffer, by Nacalai Tesque (1 mentions) Protease and phosphatase inhibitors, by Nacalai Tesque (1 mentions) Protein assay reagent kit, by Bio-Rad (1 mentions) Polyvinylidene fluoride membrane, by Merck Group (1 mentions) Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions) Flag tag (flag), by Merck Group (1 mentions) β actin, by Merck Group (1 mentions) Tris glycine gradient gel, by Bio-Rad (1 mentions)

Spelling variants of this product

Anti-AKR1C3 (4 citations) AKR1C3 (A6229, (2 citations) AKR1C3 antibody (2 citations) Anti-AKR1C3 (clone NP6.G6.A6, (2 citations)

6 protocols using akr1c3

Protein Expression Analysis by Western Blot

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Cellular protein extracts were resolved on SDS–PAGE and proteins were transferred to nitrocellulose membranes. After blocking for 1 hour at room temperature in 5% milk in PBS/0.1% Tween-20, membranes were incubated overnight at 4°C with the indicated primary antibodies [AR (441,SC-7305, Santa Cruz Biotechnology, Santa Cruz, CA); AR (N20, SC-816, Santa Cruz Biotechnology, Santa Cruz, CA) AKR1C3 (A6229, Sigma); HSD3B (SC-28206, Santa Cruz Biotechnology, Santa Cruz, CA); Tubulin (T5168, Sigma-Aldrich, St. Louis, MO)]. Tubulin was used as loading control. Following secondary antibody incubation, immunoreactive proteins were visualized with an enhanced chemiluminescence detection system (Millipore, Billerica, MA).

Liu C., Armstrong C.M., Lou W., Lombard A., Evans C.P, & Gao A.C. (2016). Inhibition of AKR1C3 activation overcomes resistance to abiraterone in advanced prostate cancer. Molecular cancer therapeutics, 16(1), 35-44.

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Immunohistochemical Analysis of Prostate Cancer

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Human prostate cancer tissues from patients undergoing radical prostectomy were used for immunohistochemical analysis. Formalin-fixed, paraffin-embedded serial tissue sections from human prostate tumor tissue specimens and LuCaP xenograft tumors were deparaffinized with xylene and rehydrated in graded EtOH. Endogenous peroxidase activity was blocked by incubating in 3% H₂O₂ for 20 min. Antigen retrieval was performed with Antigen Retrieval Citra Plus Solution (BioGenex, Freemont, CA) in a steamer. Slides were then blocked with Blocking Serum from ABC Vectastain Kit (Vector Labs, Burlingame, CA). Slides were incubated at 4°C overnight in a humidified chamber with antibodies directed against ERG (1:100; Epitomics, Burlingame, CA) or AKR1C3 (1:5000; Sigma-Aldrich, St. Louis, MO). After washing, sections were incubated with ABC Vectastain Kit, according to manufacturer's protocol, followed by incubation with 3,3-diaminobenzidine tetrahydrochloride (Vector Labs). Nuclei were counterstained with Mayer's hematoxylin (Sigma-Aldrich). Sections were then dehydrated with graded EtOH, washed with xylene, and mounted with Permount (Sigma-Aldrich).

Powell K., Semaan L., Conley-LaComb M.K., Asangani I., Wu Y.M., Ginsburg K.B., Williams J., Squire J.A., Maddipati K.R., Cher M.L, & Chinni S.R. (2015). ERG/AKR1C3/AR constitutes a feed-forward loop for AR signaling in prostate cancer cells. Clinical cancer research : an official journal of the American Association for Cancer Research, 21(11), 2569-2579.

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Immunohistochemistry of Prostate Cancer Biomarkers

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The study was approved by the Ethical Committee of the University Hospital and Faculty of Medicine and Dentistry, Palacky University in Olomouc (Ref. No. 127/14). Formalin-fixed, paraffin-embedded human prostate tumor samples were obtained after radical prostatectomies between years 1998 and 2011 and archived. Clinicopathological information is given in Table 1. Samples were immunostained with appropriate antibodies according to standard techniques: AKR1C3 (mouse monoclonal, clone NP6.G6.A6, Sigma-Aldrich; microwave antigen retrieval method in citrate buffer, pH 6.0) and HMGCS2 (rabbit monoclonal, clone EPR8642, Abcam; EnVision FLEX target retrieval method in Tris/EDTA, pH 9.0). Target expression was assessed semi-quantitatively by a pathologist using the histoscore method where the percentage of positive cells (0–100%) was multiplied by staining intensity (0–3), which resulted in a final score between 0 and 300 (H-score).

Table 1

Clinical and pathologic characteristics of patients

Parameter	Range	Tumor stage (number of patients)
Parameter		pT1–2	pT3–4	N1	Sum
Number of patients		21	26	20	67
Age (years)	49–59	8	8	7	23
	60–69	11	15	12	38
	70–75	2	3	1	6
Tumor grade (Gleason score)	≤ 6	7	3	1	11
	7	13	13	9	35
	≥ 8	1	10	10	21
Serum PSA (ng/mL)	< 4	5	2	0	7
	4–10	12	11	5	28
	> 10	4	13	15	32

Neuwirt H., Bouchal J., Kharaishvili G., Ploner C., Jöhrer K., Pitterl F., Weber A., Klocker H, & Eder I.E. (2020). Cancer-associated fibroblasts promote prostate tumor growth and progression through upregulation of cholesterol and steroid biosynthesis. Cell Communication and Signaling : CCS, 18, 11.

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Western Blot Analysis of Steroidogenic Enzymes

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Cellular protein extracts were resolved on SDS–PAGE and proteins were transferred to nitrocellulose membranes. After blocking for 1 hour at room temperature in 5% milk in PBS/0.1% Tween-20, membranes were incubated overnight at 4°C with the indicated primary antibodies [AKR1C3 (A6229, Sigma); CYP17A1 (SC-66849, Santa Cruz Biotechnology, Santa Cruz, CA); HSD3B (SC-28206, Santa Cruz Biotechnology, Santa Cruz, CA); AR (SC-815, Santa Cruz Biotechnology, Santa Cruz, CA); Tubulin (T5168, Sigma-Aldrich, St. Louis, MO)]. Tubulin was used as loading control. Following secondary antibody incubation, immunoreactive proteins were visualized with an enhanced chemiluminescence detection system (Millipore, Billerica, MA).

Liu C., Lou W., Zhu Y., Yang J.C., Nadiminty N., Gaikwad N.W., Evans C.P, & Gao A.C. (2015). Intracrine androgens and AKR1C3 activation confer resistance to enzalutamide in prostate cancer. Cancer research, 75(7), 1413-1422.

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Western Blot Analysis of Protein Expression

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For total cell lysis, the cells were washed twice with ice‐cold PBS and dissolved in Nonidet P‐40 (NP‐40) buffer containing 50 mmol/L Tris‐HCl (pH 7.5), 150 mmol/L NaCl, 0.5% NP‐40, and protease and phosphatase inhibitors (Nacalai Tesque). The protein concentration of each lysate was determined using a protein assay reagent kit (BioRad, Hercules, CA, USA). After sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, the proteins were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were then blocked for 20 min in blocking buffer (Nacalai Tesque) and probed overnight with primary antibodies for UGT2B (H‐300; detection of UGT2B family members and UGT2A1), KLK3, AR (all from Santa‐Cruz, Biotechnology, Santa Cruz, CA, USA), ACSL3, AKR1C3, FLAG and β‐actin (all from Sigma). After extensive washing, bound antibodies were visualized using HRP‐conjugated secondary antibodies and the signal was enhanced by chemiluminescence (ECL; GE Healthcare, Tokyo, Japan).

Migita T., Takayama K., Urano T., Obinata D., Ikeda K., Soga T., Takahashi S, & Inoue S. (2017). ACSL3 promotes intratumoral steroidogenesis in prostate cancer cells. Cancer Science, 108(10), 2011-2021.

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Cell Viability and Protein Expression Analysis

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Cells were seeded at a density of 10 000 cells/well in 96-well plates and were incubated in either normal media or CSS media. For pretreatment experiments, cells were treated with KV-49g or INDO for 24 h followed by the addition of ARN or ODM and incubated for a further 72 h. Cell viability was determined by the MTS tetrazolium dye assay as described previously.^{14 (link)} No intrinsic absorbance was noted with ARN, ODM, KV-49g, or INDO in the MTS assay.
Western Blotting. Cells were washed once with PBS and lysed with RIPA buffer (ThermoFisher). Proteins were quantified via BCA assay (ThermoFisher no. PI-23221) and then were resolved by SDS-PAGE on 4–20% Tris-Glycine gradient gels (BioRad). After transfer to nitrocellulose membranes, blocking was conducted for 1 h in Tris-buffered saline (10 mM Tris-HCl, 100 mM NaCl, pH 7.5) containing 0.1% Tween-20 (TBST) and 1% BSA. Samples were probed overnight at 4 °C with anti-aldo-keto reductase 1C3 enzyme (AKR1C3; Sigma; no. A6229, mouse mAb, 1:500), anti-prostate-specific antigen (PSA; Cell Signaling Technology; no. 5877S, rabbit mAb, 1:1000), or anti-β-actin (Sigma; no. A5441, mouse mAb, 1:1000), followed by incubation with anti-mouse (Sigma; no. SAB4600224) or anti-rabbit (Perkin-Elmer; no. NEF812001EA) secondary antibody (1:2000) horseradish peroxidase conjugate for 2 h, and bands were quantified by densitometry using ImageJ software.

Morsy A, & Trippier P.C. (2020). Reversal of Apalutamide and Darolutamide Aldo-Keto Reductase 1C3-Mediated Resistance by a Small Molecule Inhibitor. ACS chemical biology, 15(3), 646-650.

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