Ab124973

Manufactured by Abcam

Sourced in Germany, United States, United Kingdom

Ab124973 is a laboratory reagent used in research applications. It is a purified antibody that can be used to detect and quantify a specific target molecule. The core function of Ab124973 is to serve as a tool for researchers to investigate the presence and levels of the target analyte in their samples.

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Lab products found in correlation

Micro bca assay kit, by Thermo Fisher Scientific (1 mentions) Ab210797, by Abcam (1 mentions) Developer system, by Bio-Rad (1 mentions) Ab181602, by Abcam (1 mentions) Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions) Anti rabbit or anti mouse secondary antibody, by Santa Cruz Biotechnology (1 mentions) Ab6276, by Abcam (1 mentions) Rabbit anti lc3b, by Cell Signaling Technology (1 mentions) Rabbit anti cathepsin d, by Cell Signaling Technology (1 mentions) Goat anti eea1, by Santa Cruz Biotechnology (1 mentions) Secondary antibodies conjugated to horseradish peroxidase hrp, by GE Healthcare (1 mentions) Sc 47778 hrp, by Santa Cruz Biotechnology (1 mentions) Tris buffered saline (tbs), by Merck Group (1 mentions) Hyperfilm, by GE Healthcare (1 mentions) Ecl western blotting detection reagent, by GE Healthcare (1 mentions) Tween 20, by Merck Group (1 mentions) Complete mini, by Roche (1 mentions) Ab13995, by Abcam (1 mentions) Ab13847, by Abcam (1 mentions) Pvdf membrane, by Merck Group (1 mentions)

8 protocols using ab124973

Copper-dependent ATP7B Regulation in HepG2

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HepG2/C3a cell line and its derived clones without ATP7B expression were seeded at 1.2 × 10⁶ cells in 6-well plates (CellClear Greiner) in MEM media supplemented with 10% FBS. After overnight culture, cells were washed one time with PBS to remove cellular debris before incubation with complete medium containing Cu or BCS at the indicated concentrations for 24 h. Harvested cells were centrifuged at 5000× g for three minutes, and the pellets were washed two times with PBS before resuspension in cold RIPA cell lysis buffer (100 mM HEPES pH 7.4, 1% Triton X-100, 0.5% deoxycholate) supplemented with protease inhibitors for 20 min on ice. The cellular debris was then eliminated by centrifugation at 15,000× g for 30 min, and the supernatant fractions were analyzed by Western blot with antibodies against Atox1 (sc-100557, Santa Cruz Biotechnology, Heidelberg, Germany) and ATP7B (ab124973, abcam, Paris, France). Equal amounts of loaded proteins were previously quantified using a Micro BCA assay kit (ThermoFisher Scientific, Courtaboeuf, France).

Charbonnier P., Chovelon B., Ravelet C., Ngo T.D., Chevallet M, & Deniaud A. (2022). ATP7B-Deficient Hepatocytes Reveal the Importance of Protein Misfolding Induced at Low Copper Concentration. Cells, 11(21), 3400.

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Western Blot Protein Analysis

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Whole cell lysates were prepared and loaded onto SDS-PAGE. After transfer, the membrane was blocked with 5% skim milk for 1hr and then incubated in diluted primary antibodiesATP11B (ab05377), ATP7B (ab124973), tubulin (ab210797) or GAPDH (ab181602), obtained from Abcam, Cambridge, USA) overnight. The membrane was washed with TBS-T three times and incubated with an HRP-labeled secondary antibody for 1hr. The membrane was developed using a Bio-Rad developer system.

Hong B., Chapa V., Saini U., Modgil P., Cohn D.E., He G., Siddik Z.H., Sood A.K., Yan Y., Karuppaiyah S., Pei G., Zhao Z., Yoo J.Y, & Kaur B. (2020). Oncolytic HSV therapy modulates vesicular trafficking inducing cisplatin sensitivity and anti-tumor immunity. Clinical cancer research : an official journal of the American Association for Cancer Research, 27(2), 542-553.

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Western Blot Analysis of ATP7B

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Transfected HEK293 cells were lysed in RIPA buffer (0.05 M tris-HCl, pH 7.0; 0.15 M NaCl; 0.25 % deoxycholic acid; 1% NP-40; 1 mM EDTA; Millipore) for 30 min on ice. Centrifugation at 3,000 g for 15 min removed cell debris. The supernatant was collected and used for further studies. Protein concentration was determined using BCA assay (Pierce). Thirty μg of protein were separated on 10% Laemmli gels and then transferred to PVDF (Millipore) membranes using CAPS, pH 11 transfer buffer. Membranes were blocked for 1 h at room temperature (RT) in 5% milk in phosphate-buffered saline (PBS), then incubated for overnight (16 h) in either 1:6000 anti-ATP7B (Abcam, ab124973) or 1:2000 anti-β-actin (Abcam, ab6276) diluted in PBS containing 0.2% Tween-20 (PBST) with 0.05% sodium azide. Membranes were then incubated for 1 h at room temperature (RT) in either 1:10,000 anti-mouse or anti-rabbit secondary antibodies (Santa Cruz) before imaging on an Alpha-Imager (ProteinSimple). The fluorescent intensities of protein bands were determined with Image-J (NIH) and ATP7B levels were normalized to a β-actin loading control.

McCann C.J., Jayakanthan S., Siotto M., Yang N., Osipova M., Squitti R, & Lutsenko S. (2019). SINGLE NUCLEOTIDE POLYMORPHISMS IN HUMAN ATP7B GENE MODIFY PROPERTIES OF ATP7B PROTEIN. Metallomics : integrated biometal science, 11(6), 1128-1139.

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Immunostaining Protocol for ATP7B in WIF-B Cells

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For experiments that used the rat anti-ATP7B antibody, WIF-B cells were processed at room temperature as follows. Cells were briefly rinsed in PBS, fixed 30 minutes in 4% paraformaldehyde/PBS then permeabilized for 5 minutes in 0.2%Triton-X 100 in PHEM buffer (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, and 2 mM MgCl₂, pH 6.8). Fixed and permeabilized cells were incubated for 30 minutes in blocking buffer (PBS containing 1% BSA and 0.1% Triton-X100), and incubated in primary antibodies (1 hour) and secondary antibodies (30 minutes) diluted in blocking buffer. For experiments that employed the rabbit polyconal ATP7B (#ab124973, Abcam) cells were fixed on ice and permeabilized as previously described (22 (link)).

Nyasae L.K., Schell M.J, & Hubbard A.L. (2014). Copper Directs ATP7B to the Apical Domain of Hepatic Cells via Basolateral Endosomes. Traffic (Copenhagen, Denmark), 15(12), 1344-1365.

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Antibody Immunofluorescence Assay Protocol

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Primary antibodies used for indirect immunofluorescence were as follows: for Figures 1, 4, S5, and 6 we used rat anti-ATP7B (S. Lutsenko, Johns Hopkins School of Medicine); For Figures 2, S3, S4, 5, 7 & 8 we used rabbit anti-ATP7B (#ab124973, Abcam, Cambridge, MA); for Figures 3 and S1 we used rabbit anti-ATP7B (Dr. J. Gitlin, Brown Alpert Medical School, Providence, RI); rabbit anti-aminopeptidase N (APN, #1637, (22 (link)) guinea pig anti-dipeptidyl-peptidase 4 (DPP4, (41 (link))) goat anti-EEA1 (#sc-6415, Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-TGN38, and -Syntaxin 6 (BD Biosciences, San Jose, CA); mouse anti-LAMP1 (#H4A3-s, Developmental Studies Hybridoma Bank, Iowa City, IA); rabbit anti-Cathepsin D (Dr A. Hasilik, Marburg, Germany); rabbit anti-LC3B (#2775, Cell Signaling Technology, Danvers, MA). Secondary antibodies conjugated to Cy3 or Cy5 were from Jackson ImmunoResearch Laboratories (West Grove, PA), while those conjugated to Alexa 488, -568 or -647 were from Molecular Probes (Eugene, OR). Primary antibodies used for immunoblotting inculded: rabbit anti-ATP7B (#3985) (13 (link)), mouse anti-alpha tubulin, DM1A (Sigma, St. Louis, MO). Secondary antibodies conjugated to horseradish peroxidase (HRP) were from G.E. Healthcare (Buckinghamshire, United Kingdom).

Nyasae L.K., Schell M.J, & Hubbard A.L. (2014). Copper Directs ATP7B to the Apical Domain of Hepatic Cells via Basolateral Endosomes. Traffic (Copenhagen, Denmark), 15(12), 1344-1365.

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Western Blot Analysis of ATP7B Protein

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Cells were homogenized in RIPA lysis buffer (60 mM tris-HCl, 150 mM NaCl, 2% Na-deoxycholate, 2% Triton X-10, 0.2% SDS, and 15 mM EDTA) and protease inhibitors (Roche, Basel, Switzerland; Complete Mini, EDTA-free). 10 μg protein lysate of whole protein extract was separated on a 10% SDS gel. Samples were blotted onto PVDF membranes and blocked with 5% semi-skimmed milk powder (Roth, Karlsruhe, Germany) in Tris-buffered saline supplemented with 0.1% Tween 20 (Merck, Darmstadt, Germany). For detection of ATP7B protein (ATPase7), a monoclonal rabbit anti-human ATP7B antibody (1:1,000, ab124973, Abcam, Cambridge, UK) was added overnight. β-Actin was assessed for protein loading control (1:1,000, sc-47778 HRP, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary antibody was horseradish peroxidase-conjugated anti-rabbit (1:10,000; GE Healthcare life science, München, Germany) and incubated 2 hours at room temperature. For detection, ECL Western Blotting Detection Reagent (GE Healthcare) was added and the membrane was exposed to film (Hyperfilm, GE Healthcare).

Pöhler M., Guttmann S., Nadzemova O., Lenders M., Brand E., Zibert A., Schmidt H.H, & Sandfort V. (2020). CRISPR/Cas9-mediated correction of mutated copper transporter ATP7B. PLoS ONE, 15(9), e0239411.

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Protein Expression Analysis Protocol

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The cells were lysed, and total proteins were extracted and examined for concentration using BCA Protein Assay Kit. Cell lysates were separated by 12% SDS‐PAGE and transferred onto PVDF membranes (Millipore, CA). These PVDF membranes were incubated with the antibodies against Caspase‐3 (Abcam, 1:1000, ab13847), ATP7A (Abcam, 1:1000, ab13995), ATP7B (Abcam, 1:1000, ab124973), and β‐actin (Abcam, 1:1000, ab8227) overnight at 4°C and further incubated with HRP‐conjugated secondary antibody (1:6000).

Yu Z., Cao W., Ren Y., Zhang Q, & Liu J. (2020). ATPase copper transporter A, negatively regulated by miR‐148a‐3p, contributes to cisplatin resistance in breast cancer cells. Clinical and Translational Medicine, 10(1), 57-73.

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Subcellular Localization of ATP7B

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HepG2 cells were plated at 150,000 cells per well on acid-washed and sterilized glass coverslips in a 12-well plate. Cells were stimulated as described before and then washed at 1, 6, 12, and 24 h in cold PBS (Gibco) and fixed for 10 min in 4% paraformaldehyde (AAJ19943K2, Thermo Fisher Scientific). Cells were blocked in 10% BSA and permeabilized with Triton X-100. 1:600 anti-ATP7B (ab124973, Abcam) and 1:600 anti-TGN46 (GTX74290, GeneTex) antibodies were used to stain the copper transporter and trans-Golgi network marker, respectively, and anti-rabbit IgG AlexaFluor 488 (R37116, Invitrogen) and anti-sheep IgG AlexaFluor 647 (A21448, Invitrogen) were used as secondary antibodies to fluorescently label the proteins. A DAPI dye (R37606, Invitrogen) was used to stain the nucleus, and coverslips were sealed to glass slides along with Prolong Gold Antifade Mountant (P36930, Invitrogen). Fixed and mounted cells were imaged using a laser scanning confocal microscope (Olympus FluoView FV1000) using a ×60 oil immersion lens at the UC Davis Molecular and Cellular Biology Light Microscopy Imaging Facility.

Harder N.H., Lee H.P., Flood V.J., San Juan J.A., Gillette S.K, & Heffern M.C. (2022). Fatty Acid Uptake in Liver Hepatocytes Induces Relocalization and Sequestration of Intracellular Copper. Frontiers in Molecular Biosciences, 9, 863296.

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