For western blot, total muscle lysates were prepared in a buffer containing 50 mM Tris, 100 mM NaCl, 1 mM EGTA, 0.5% NP-40, 0.5% Triton-X100, 0.1% SDS, 1 mM DTT, 1 mM PMSF, and a mix of protease inhibitor (Complete EDTA-free, Roche, Basel, Switzerland). For human muscles, 50 μg of protein extracts were loaded on a 10% SDS-Page gel. For murine muscles, 5 μg of protein extracts were loaded on 4–15% Mini-PROTEAN®TGX™ precast protein gel (Biorad, Hercules, USA). Following primary and secondary antibodies were used: rabbit anti-alpha-actinin-2 (ab68167, Abcam), rabbit anti-alpha-actinin-3 (ab68204, Abcam), mouse anti-actin (homemade), mouse anti-beta-tubulin (homemade), mouse anti-GAPDH (MAB374, Millipore, Burlington, USA), and horseradish peroxidase (Jackson immunoresearch Europe, Cambridgeshire, UK). Membranes were revealed with the Supersignal west pico kit (ThermoFisher Scientific), and all immunoblots were visualized on an Amersham Imager 600 (GE Healthcare Life Sciences, Chicago, USA).
Ab68167
Manufactured by Abcam
Sourced in United Kingdom
Ab68167 is a monoclonal antibody that recognizes the protein marker HER2/neu. It is intended for use in immunohistochemistry (IHC) applications to detect the presence and distribution of HER2/neu in tissue samples.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 or 555, by Thermo Fisher Scientific (2 mentions)
Anti phospho histone h3 ser10, by Merck Group (2 mentions)
Sc 47739, by Merck Group (2 mentions)
Anti phospho histone gamma h2ax, by Merck Group (2 mentions)
Ab62623, by Abcam (2 mentions)
Antifade mounting medium, by Vector Laboratories (2 mentions)
Epitope retrieval solution, by IHC World (2 mentions)
Anti aurora b, by Merck Group (2 mentions)
Ab18061, by Abcam (2 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
Supersignal west pico kit, by Thermo Fisher Scientific (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Complete edta free, by Roche (1 mentions)
Mouse anti gapdh mab374, by Merck Group (1 mentions)
Horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Amersham imager, by Cytiva (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
Anti phospho mtor, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
14 protocols using ab68167
1
Optimized Western Blot Protocol
2
Cordycepin Modulates AMPK and mTOR Pathways
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Cordycepin specified to be over 99.2% pure as determined by HPLC was purchased from Shanghai Winherb Medical Co. (Shanghai, China). Primary antibodies against the following proteins were obtained from Cell Signalling Technology (Danvers, MA): anti‐AMPKα (#2603P), anti‐phospho‐AMPKα (#2535), anti‐mammalian target of rapamycin (mTOR) (#2983), anti‐phospho‐ mTOR (#2971), anti‐phospho‐ERK, #4370P), anti‐ERK (#4695), anti‐acetyl‐CoA carboxylase (ACC) (#3676), anti‐phospho‐ACC (#3661), and anti‐GAPDH (#2118). Primary antibodies against gp91phox (ab129068), superoxide dismutase 1 (SOD1, ab16831), SOD2 (ab38155), and α‐actinin (ab68167) were purchased from Abcam (Cambridge, UK). The secondary antibody was purchased from LI‐COR Biosciences. Ang II (A9525) and compound C (CpC, P5499) were purchased from Sigma‐Aldrich. Proteins were measured with assay kits obtained from Pierce (Pierce, 23225).
3
Immunohistochemistry of Cardiac Tissues
4
Quantifying Dystrophin and Alpha-Actinin
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Muscles were dissected and frozen in liquid-nitrogen cooled isopentane. 8μm sections were lysed in High SDS buffer containing 0.02% EDTA (pH 8.0), 0.075% Tris-HCl (pH 6.8), and protease and phosphatase inhibitors. Capillary Western immunoassay (Wes) analysis was performed according to manufacturer’s instructions using 66–440 kDa Separation Modules (ProteinSimple). In each capillary 0.2mg/mL protein was loaded for analysis with antibodies to dystrophin (Abcam #ab15277, dilution 1:15) or alpha-actinin (Abcam #ab68167, dilution 1:100), and anti-rabbit secondary (ProteinSimple #042-206). Compass for SW software was used to quantify chemiluminescence data, with data reported as % bmx + vehicle.
5
Immunofluorescence Staining of Sarcomeric α-Actinin
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Cells grown on glass coverslips (Thermo Scientific) were fixed with −20 °C methanol (JT Baker) for 5 min, permeabilized for 2 h with 10% normal goat serum (Thermo Fisher) and 0.3% Triton X-100 in PBS. After treatment with blocking buffer (1% BSA in PBS/0.3% Triton X-100), immunofluorescence staining of the cells was performed by incubation with primary anti-sarcomeric α-actinin antibody (Abcam, ab68167) diluted in 1% BSA in PBS/0.3% Triton X-100, washing in PBS, and incubation for 1 h with Cy5-labeled secondary antibody (Invitrogen A10523). Images were obtained with an inverted confocal laser-scanning microscope (Zeiss).
6
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 ).
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7
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).
8
Immunofluorescence Analysis of Apoptosis and Angiogenesis
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Immunofluorescence staining was performed on deparaffinized sections. After dewaxed and hydrated, the nonspecific antigens of the specimens were blocked with 3% H2O2 for 15 min and with 5% bovine serum albumin (BSA) (Beyotime) in PBS for 45 min at 37 °C, respectively. Subsequently, the sections were incubated with primary anti-Cas-3 (ab32351, 1:200, Abcam) and anti-α-sarcomeric actin (ab68167, 1:200, Abcam) antibodies at 4 °C overnight followed by goat anti-rabbit IgG Alexa FluorVR 647 (ab150083, 1:1500, Abcam, Cambridge, United Kingdom). Nuclei were stained using DAPI (Beyotime, China). Angiogenesis was observed using a fluorescence microscope (Nikon, Japan).
9
Immunohistochemistry of Cardiac Tissues
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Tissues were fixed in 4% paraformaldehyde in PBS overnight at 4°C
and incubated in 30% sucrose in PBS at 4°C overnight. Tissues were
embedded in tissue freezing medium and cut 8 μm thicknesses. For antigen
retrieval, either 1 mM EDTA with 0.05% Tween 20 in boiling water or epitope
retrieval solution (IHC World) in a steamer (IHC-Tek Epitope Retrieval Streamer
Set) were used, then sections were blocked with 10% serum from the host animal
of secondary antibodies, and incubated with primary anti-bodies overnight at 4
°C. The sections were subsequently washed with PBS and incubated with
corresponding secondary antibodies conjugated to Alexa Fluor 488 or 555
(Invitrogen). The slides were mounted in antifade mounting medium (Vector
Laboratories, Burlingame, California). Primary antibodies used are as follows:
anti-phospho histone H3 Ser10 (Millipore 06–570, 1:100), anti-aurora B
(Sigma A5102, 1:100), anti-troponin T, cardiac isoform Ab-1, clone 13–11
(Thermo Scientific MS-295-P1, 1:100), anti-sarcomeric α-actinin (Abcam,
ab68167, 1:100), anti-8 hydroxyguanosine (Abcam ab62623, 1:50),
anti-phosphorylated ATM (Santa Cruz Biotechnology sc-47739, 1:100),
Anti-phospho-Histone gamma H2AX (Millipore-Sigma 05–636). DAPI was used
for the nuclear staining.
and incubated in 30% sucrose in PBS at 4°C overnight. Tissues were
embedded in tissue freezing medium and cut 8 μm thicknesses. For antigen
retrieval, either 1 mM EDTA with 0.05% Tween 20 in boiling water or epitope
retrieval solution (IHC World) in a steamer (IHC-Tek Epitope Retrieval Streamer
Set) were used, then sections were blocked with 10% serum from the host animal
of secondary antibodies, and incubated with primary anti-bodies overnight at 4
°C. The sections were subsequently washed with PBS and incubated with
corresponding secondary antibodies conjugated to Alexa Fluor 488 or 555
(Invitrogen). The slides were mounted in antifade mounting medium (Vector
Laboratories, Burlingame, California). Primary antibodies used are as follows:
anti-phospho histone H3 Ser10 (Millipore 06–570, 1:100), anti-aurora B
(Sigma A5102, 1:100), anti-troponin T, cardiac isoform Ab-1, clone 13–11
(Thermo Scientific MS-295-P1, 1:100), anti-sarcomeric α-actinin (Abcam,
ab68167, 1:100), anti-8 hydroxyguanosine (Abcam ab62623, 1:50),
anti-phosphorylated ATM (Santa Cruz Biotechnology sc-47739, 1:100),
Anti-phospho-Histone gamma H2AX (Millipore-Sigma 05–636). DAPI was used
for the nuclear staining.
10
Antibody Dilutions for Western Blotting and Immunofluorescence
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Anti-SIRT1 antibodies [human specific: 2493, 1:1000 dilution for western blotting (WB); mouse specific: 2028, 1:1000 dilution for WB] were from Cell Signaling Technology. Anti-ACTN2 antibodies [ab9465, 1:50 dilution for immunofluorescence (IF); ab68167, 1:1000 dilution for WB and 1:200 for IF] and anti-Na+/K+ ATPase antibody (ab76020, 1:200 dilution for IF) were from Abcam. Anti-Actin antibody was from Millipore (MAB1501, 1:10000 dilution for WB). Anti-CCL2 antibody (MABN712; 1:200 dilution for IF) was from Sigma, anti-CCR4 antibody (MAB1567; 1:200 dilution for IF) was from R&D Systems, and anti-CCL12 antibody (abx103190; 1:200 dilution for IF) was from Abbexa. In ChIP analysis, 3 μl anti-histone H3 (acetyl K27) antibody (Abcam, ab4729) was used per reaction.
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