Ab18061

Manufactured by Abcam

Ab18061 is a primary antibody developed by Abcam. It is a rabbit polyclonal antibody that recognizes a specific epitope. The core function of this product is to bind to and detect the target antigen in various applications, such as Western Blot, Immunohistochemistry, and Immunocytochemistry.

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Lab products found in correlation

Ab45932, by Abcam (2 mentions) Ab68167, by Abcam (2 mentions) Lsm 710, by Zeiss (2 mentions) Ab71333, by Abcam (1 mentions) Rab11, by Thermo Fisher Scientific (1 mentions) H9658, by Cell Signaling Technology (1 mentions) E cadherin, by Cell Signaling Technology (1 mentions) Alexa fluor 488 or 594 secondary antibody, by Thermo Fisher Scientific (1 mentions) Mab4360, by Merck Group (1 mentions) Ma1 83347, by Thermo Fisher Scientific (1 mentions) Eclipse 80i fluorescent microscope, by Nikon (1 mentions) Hoechst 33342, by Thermo Fisher Scientific (1 mentions) Bz x700, by Keyence (1 mentions) Ab28258, by Abcam (1 mentions) Ab156002, by Abcam (1 mentions) Sds page, by Thermo Fisher Scientific (1 mentions) Prism 6, by GraphPad (1 mentions) Chemidoc imaging system, by Bio-Rad (1 mentions) Ab32572, by Abcam (1 mentions) Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)

9 protocols using ab18061

Detailed Western Blot and Immunofluorescence Protocols for Huntingtin Protein

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For western blot: polyclonal anti-Huntingtin 1–17 Ab1(1μg/ml) [39 (link)], α-actinin-2 (1:2000; ab68167; Abcam; specific [37 (link)], α-actinin (2μg/ml; ab18061; Abcam; may cross react with isoforms 1–4), GAPDH (1:6000, MAB374, Millipore). For fluorescent labeling: Anti-Huntingtin Ab2527 and Ab1173 were previously described [40 (link), 41 (link)]. Both are rabbit polyclonal made against peptides covering aa 2527–2547 and aa 1173–1196 of Huntingtin, respectively and were used at 3 μg/ml. The immunogen peptides consisted of the following amino acids: “ySCLEQQPRNKPLKALDTRFGR” and “ySLTNPPSLSPIRRKGKEKEPGEQA”. Anti-α-actinin (1:500; ab18061; Abcam; may cross react with isoforms 1–4), vinculin (1:100; Sigma). Secondary antibodies include Cy3 goat anti-mouse secondary antibody (1:500; Jackson Immunoresearch); Cy3 goat anti-rabbit secondary antibody (1:500; Jackson Immunoresearch); Bodipy Green goat anti-mouse secondary antibody (1:500; Invitrogen). Stains include Alexa Fluor 488-Phalloidin and Rhodamine-Phalloidin (1:500; Molecular Probes) for F-actin, and DAPI (1:500; Sigma) or Hoechst stain (1:1000; Molecular Probes) for nuclei. For peptide blocking experiments, 3 μg/ml Ab2527 in 4% Normal Goat Serum/PBS was incubated overnight with 30 μg/ml peptide (above) overnight at 4°C or with an unrelated peptide (yEPGDQENKPCRIKGDIGQST).

Tousley A., Iuliano M., Weisman E., Sapp E., Richardson H., Vodicka P., Alexander J., Aronin N., DiFiglia M, & Kegel-Gleason K.B. (2019). Huntingtin associates with the actin cytoskeleton and α-actinin isoforms to influence stimulus dependent morphology changes. PLoS ONE, 14(2), e0212337.

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Immunofluorescence Analysis of Cell Adhesion

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Antibodies to paxillin (Abcam, ab2264, rabbit poly Ab), phalloidin-TRITC (Sigma, P1951), tubulin (Millipore, MAB1864, rat mAb), actinin (Abcam, ab18061, mouse mAb) talin (Abcam, ab71333, rabbit poly Ab), β1 integrin (Abcam, clone 12G10, ab30394, mouse mAb; and BD Biosciences, clone 9EG7, 553715, rat mAb), MB1.2 (Millipore, MAB1997, rat mAb), Rab5 (Cell Signaling Technology, 3547, rabbit mAb), Rab11 (Thermo Fisher Scientific, 71–5300, rabbit polyAb), Rab7 (Thermo Fisher Scientific, PA5–22959, rabbit polyAb), IQGAP1 (BD Biosciences, 610612, mouse mAb; and Abcam, ab86064, rabbit poly Ab), ARF6 (Cell Signaling Technology, 3546, rabbit poly Ab), HA (Sigma, H9658, mouse mAb), E-cadherin (Cell Signaling Technology, 3195, rabbit mAb), GAPDH-peroxidase; (Sigma, G9295, mouse mAb), EFA6 (Atlas Antibodies, HPA059237, rabbit poly Ab), EFA6 (Thermo Fisher Scientific, PA5–31153, rabbit poly Ab), and ARNO (Sigma, SAB2500109, goat poly Ab) were purchased, as stated for each above.

Ghosh M., Lo R., Ivic I., Aguilera B., Qendro V., Devarakonda C, & Shapiro L.H. (2019). CD13 tethers the IQGAP1-ARF6-EFA6 complex to the plasma membrane to promote ARF6 activation, B1 integrin recycling, and cell migration. Science signaling, 12(579), eaav5938.

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Immunofluorescence Staining of Pluripotent and Cardiac Cells

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Pluripotent stem cells or differentiated cardiomyocytes were grown on coverslips and fixed in 4% paraformaldehyde at room temperature for 10 min, then blocked with 5% goat serum in PBS and 0.1% Tween 20 for one hr at room temperature. Fixed cells were stained with SSEA4 antibody (clone MC-813–70, R&D systems), OCT3/4 antibody (sc-9081, Santa Cruz Biotechnology), NANOG antibody (sc-33759, Santa Cruz Biotechnology), TRA-1–60 antibody (MAB4360, Millipore), Hoechst 33342 (Life Technologies), cardiac troponin T antibody (ab45932, Abcam), cardiac myosin antibody (MA1–83347, Thermo Fisher), or alpha-actinin antibody (ab18061, Abcam) at 4°C overnight. The sections were incubated with Alexa Fluor 488 or 594 secondary antibodies (Life Technologies) to visualize the specific stains. Image acquisition was performed on an Eclipse 80i fluorescent microscope (Nikon instruments, New York, USA), Keyence BZ-X700 (Keyence Corporation, Osaka, Japan), or confocal microscope (Zeiss LSM 710).

Wilson K.D., Ameen M., Guo H., Abilez O.J., Tian L., Mumbach M.R., Diecke S., Qin X., Liu Y., Yang H., Ma N., Gaddam S., Cunningham N., Gu M., Neofytou E., Prado M., Hildebrandt T.B., Karakikes I., Chang H.Y, & Wu J.C. (2020). Endogenous Retrovirus-Derived lncRNA BANCR Promotes Cardiomyocyte Migration in Humans and Non-human Primates. Developmental cell, 54(6), 694-709.e9.

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Quantification of Neuromuscular Junction Proteins

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Proteins were fractionated by SDS-PAGE (6% or 8% acrylamide; Invitrogen) and transferred to polyvinylidene fluoride membranes. Blots were probed with antibodies to the AChR β-subunit (N8283, RRID:AB_262151; Sigma-Aldrich), Rapsyn (ab156002, RRID:AB_298028; Abcam), MACF1 (gift from R. Liem, Columbia University Medical Center, New York, NY; Chen et al., 2006 (link)), β-Catenin (ab32572, RRID:AB_725966; Abcam), α-Actinin (ab18061, RRID:AB_444218; Abcam), Calpain (ab28258, RRID:AB_725819; Abcam), or MAP1b (ab11266, RRID:AB_297884; Abcam). We used antibodies to the AChR β-subunit to ensure equal loading of proteins in whole-cell lysates and proteins isolated with biotin–α-BGT. We quantified the band intensities with a ChemiDoc imaging system (BioRad), as described previously (Remédio et al., 2016 (link)). The band intensity for each protein was indexed to the band intensity of the AChR β-subunit, and the graphs show the mean values from three separate experiments. The Wilcoxon–Mann–Whitney test was used to determine statistical significance and was conducted using GraphPad Prism 6.0 software.

Oury J., Liu Y., Töpf A., Todorovic S., Hoedt E., Preethish-Kumar V., Neubert T.A., Lin W., Lochmüller H, & Burden S.J. (2019). MACF1 links Rapsyn to microtubule- and actin-binding proteins to maintain neuromuscular synapses. The Journal of Cell Biology, 218(5), 1686-1705.

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Immunoblotting Markers for Huntington's Disease

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Polyclonal anti-htt 1–17 Ab1(1μg/ml) [42 (link)], mAb 1C2 to polyglutamines (MAB1574, 1:1000; Millipore), mAb 3B5H10 to polyglutamines (P1874, 1:10,000; Sigma), p85 PI 3-kinase (1:500; #4292, Cell Signaling), AKT and phospho (Ser473) AKT (1:500; Cell Signaling); ERK and phospho ERK (1:1000; Cell Signaling); β-tubulin (1:4000; T8328, Sigma), beta3-tubulin (1:2000; Sigma), anti-GFAP (1:2000; Millipore), Rac1 (1:2000; Millipore), nestin (1:500; Millipore), DARPP32 (1:5000; Abcam), Islet1 (1:200; University of Iowa Developmental Studies Hybridoma Bank), GAPDH (1:6000; Millipore), α-actinin-2 (1:250; mAb clone EA-53, Sigma) or α-actinin-2 (1:2000; ab68167; Abcam; [31 (link)], α-actinin (2μg/ml; ab18061; Abcam; may cross react with isoforms 1–4).

Tousley A., Iuliano M., Weisman E., Sapp E., Zhang N., Vodicka P., Alexander J., Aviolat H., Gatune L., Reeves P., Li X., Khvorova A., Ellerby L.M., Aronin N., DiFiglia M, & Kegel-Gleason K.B. (2019). Rac1 Activity Is Modulated by Huntingtin and Dysregulated in Models of Huntington’s Disease. Journal of Huntington's Disease, 8(1), 53-69.

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Immunolabeling of Neuronal Cytoskeleton

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Neurons were fixed with pre-warmed 4 % paraformaldehyde/4 % sucrose in PBS for 15 min, permeabilized with 0.1 % Triton X-100 in PBS for 10 min, and blocked with a blocker solution (2 % bovine serum albumin, 2 % fetal bovine serum, and 0.2 % fish gelatin in PBS) for 1 h at room temperature, as described previously [4 (link)]. Cells were then incubated the primary antibodies, including a mouse monoclonal anti-α-tubulin antibody (1:1000, Sigma, no. T-5168), a goat polycolonal anti-TRPM7 (1:200, Abcam, Ab729), a mouse monoclonal anti-α actinin (1:500, Abcam, Ab18061), or a mouse monoclonal anti-NeuN (1:500, Millipore, no. MAB377). To differentiate between axons and dendrites, neurons were incubated with mouse monoclonal anti-tau-1 (axonal marker; 1:500, Millipore, MAB3420) and chicken polyclonal anti-microtubule associated protein 2 (MAP2) (dendritic marker; 1:500, Millipore, Ab15452).

Turlova E., Bae C.Y., Deurloo M., Chen W., Barszczyk A., Horgen F.D., Fleig A., Feng Z.P, & Sun H.S. (2014). TRPM7 Regulates Axonal Outgrowth and Maturation of Primary Hippocampal Neurons. Molecular neurobiology, 53(1), 595-610.

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Cytoskeletal Protein Immunostaining Protocol

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Forskolin (F6886), epinephrine (E4642), phorbol-12-myristate-13-acetate (P1585), heparin (H3149), 4′, 6-diamidino-2 phenylindole dihydrochloride (DAPI, D8417), dimethylsulfoxide (D2650) and anti-flag M2 affinity gel (A2220) were from Sigma. Rabbit anti-VWF antibody (A0082) was from Dako. Rabbit polyclonal antibody to ERK2 (sc-292838) was from Santa Cruz Biotechnology. Rabbit and mouse monoclonal antibodies against zyxin (ab109316, ab50391), mouse monoclonal antibodies to vinculin (ab18058) and mouse monoclonal antibody to mouse α-actinin (ab18061) were from Abcam. Mouse monoclonal antibody to human P-selectin (Q01102) was from R&D Systems. Rabbit polyclonal antibody to phospho-zyxin Ser 142/143 (#4863) was from Cell Signaling Technology. Alexa Fluor 488-nm-conjugated phalloidin (A12379) and Alexa Fluor-conjugated goat anti-IgG antibodies (A11008, A27039, A21424, A21052) were from Invitrogen.

Han X., Li P., Yang Z., Huang X., Wei G., Sun Y., Kang X., Hu X., Deng Q., Chen L., He A., Huo Y., Li D., Betzig E, & Luo J. (2017). Zyxin regulates endothelial von Willebrand factor secretion by reorganizing actin filaments around exocytic granules. Nature Communications, 8, 14639.

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iPSC-derived Cardiomyocyte Immunostaining

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Patient‐specific iPSC‐derived cardiomyocytes maintained on iMatrix‐coated 35‐mm glass bottom dishes (Matsunami Glass Ind.) were stained with the following primary Abs: rabbit anti‐cTnT (ab45932; Abcam) and mouse anti‐α‐actinin (ab18061; Abcam). Alexa Fluor 488 goat anti‐rabbit or Alexa Fluor 594 goat anti‐mouse Abs (Thermo Fisher Scientific) were used as secondary Abs.

Sasaki R., Kurebayashi N., Eguchi H., Horimoto Y., Shiga T., Miyazaki S., Kashiyama T., Akamatsu W, & Saito M. (2022). Involvement of kallikrein‐PAR2‐proinflammatory pathway in severe trastuzumab‐induced cardiotoxicity. Cancer Science, 113(10), 3449-3462.

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Focal Adhesion Turnover Analysis

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HFFs were plated in SFM for 2 hours on coverslips pre-coated with FN or
cFN. For negative controls of FA turnover, cells were plated on FN in the
presence of 10uM PF-28 for the full 2 hours or 2uM of cytochalasin D for the
last 30 minutes of cell plating. After 2 hours, cells were washed, fixed in
paraformaldehyde, and stained with anti-α-actinin (ab18061, Abcam) at
1:400, anti- rabbit paxillin (ab32084, abcam), and phalloidin. Only individual
cells determined not to be in contact with any neighboring cells were imaged via
PerkinElmer spinning disk confocal microscope using a 63X objective. A total of
75 cells over 4 coverslips were imaged per condition. Three biological
replicates were performed, only one of which included sh-RNA β1 knockdown
cells on FN or cFN as additional groups.

Stefanelli V.L., Choudhury S., Hu P., Liu Y., Schwenzer A., Yeh C.R., Chambers D.M., von Beck K., Li W., Segura T., Midwood K.S., Torres M, & Barker T.H. (2019). Citrullination of Fibronectin alters integrin clustering and focal adhesion stability promoting stromal cell invasion. Matrix biology : journal of the International Society for Matrix Biology, 82, 86-104.

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