The following antibodies were used: anti-FLAG (Sigma, Cat: F3165, dilution 1:10,000), anti-Myc (Abcam, Ab9106, 1:2500), anti-Phosphothreonine (pThr, Thermo Fisher, 71-8200, 1:2500); anti-Phosphoserine (pSer, Thermo Fisher, 61–8100, 1:2500); anti-LETM1 (Santa Cruz, mouse sc-514136, 1:2500 or Novus Biologicals, rabbit NBP1-33433, 1:10,000). Anti-PINK1 (Abgent, mouse AM6406a, 1:5000 and Novus Biologicals, rabbit BC100–494, 1:5000), anti-Tyrosine hydroxylase (TH, EMD Millipore, AB152, 1:1000), anti-MCU (Cell Signaling, 14997, 1:10,000), anti-NCLX (Abcam, ab136975, 1:5000). Special custom phospho-LETM1 at Thr192 (pT192) polyclonal antibody was generated and purified from a rabbit immunized with carrier protein-conjugated phosphopeptide, CNGHTLpT192RRERR (residues 187–197 of human LETM1, NP_036450.1), using standard protocols by Biogenes (Berlin, Germany, dilution 1:2500).
Ab152
Manufactured by Cell Signaling Technology
AB152 is a primary antibody used in immunohistochemistry and Western blotting applications. It targets a specific protein and can be used to detect and analyze the presence and distribution of that protein in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Dapi fluoromount g, by Southern Biotech (2 mentions)
Vt1000, by Leica (2 mentions)
Af5869, by Abcam (2 mentions)
Ab18181, by Synaptic Systems (2 mentions)
Anti flag, by Merck Group (1 mentions)
Anti mcu, by Cell Signaling Technology (1 mentions)
Bc100 494, by Merck Group (1 mentions)
Ab9106, by Abcam (1 mentions)
Anti myc, by Abcam (1 mentions)
Iblot2 transfer system, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Gradient gel, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Bullet blender bead lysis kit, by Next Advance (1 mentions)
Ecl detection kit, by Thermo Fisher Scientific (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Cofilin 1, by Cell Signaling Technology (1 mentions)
Gapdh, by Proteintech (1 mentions)
Mab1501, by Merck Group (1 mentions)
7 protocols using ab152
1
Comprehensive Antibody Profiling Protocol
2
Immunofluorescence Staining of Mouse Brain
3
Protein Extraction and Expression Analysis
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Proteins were extracted from frozen hearts and soleus muscle explants. Tissues were submerged in ice-cold RIPA buffer (ThermoFisher, #89900) containing protease and phosphatase inhibitors (ThermoFisher, #A32959). Tissues were powdered and homogenized using a bullet blender bead lysis kit (Next Advance). The samples were spun at 800 g for 2 min at 4°C and supernatant was collected and centrifuged at 12,000 g for 10 min at 4°C. Protein concentrations were determined using a Pierce BCA Protein Assay Kit (ThermoFisher, #23225). Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 4%–20% gradient gels (Bio-Rad, #4561094) and then transferred to polyvinylidene fluoride (PVDF) membranes (ThermoFisher, #IB24002) using the iBlot 2 transfer system (ThermoFisher). Protein expression was measured by chemiluminescence using the ChemiDoc imaging system (Bio-Rad). Protein expression was quantified using Ponceau S (ThermoFisher, #A40000279). The following primary antibodies were used: Tyrosine hydroxylase, 1:1000 (Millipore Ab152), AKT, 1:1000 (Cell Signal 9272s), pAKT, 1:1000 (Cell Signal 9271s), ChAT, (1:2000, ab178850).
4
Protein Expression Analysis by Western Blot
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PI protein extracts (15 µg) were solubilized in SDS-PAGE buffer (62.5 mM Tris pH 6.8, 2% SDS, 10% glycerol, 0.00125% bromophenol blue, and 15% β-mercaptoethanol). The extracts were boiled for 5 min, separated (15 µg) on 10% SDS-PAGE electrophoresis gel and transferred onto a nitrocellulose membrane. All membranes were blocked and incubated with an appropriate dilution of the primary antibody: TH antibody (1:2000; Millipore AB152) and β-actin (1:1000; Cell Signaling #3700). Membranes were next washed and incubated with a horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG secondary antibody (1: 2500; (Cell Signaling) followed by chemiluminescent detection, using an enhanced chemiluminescence (ECL) detection kit (ThermoFisher Scientific). Films were scanned and the density of each band was measured with Image J software. TH expression levels were normalized to β-actin.
5
Protein Expression Analysis in Mouse Brain
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The mouse brain tissue samples were lysed in lysis buffer (50 mM Tris, pH 7.4, 40 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 1.5 mM Na3VO4, 50 mM NaF, 10 mM sodium pyrophosphate and 10 mM sodium β-glycerophosphate, supplemented with protease inhibitors cocktail), and centrifuged for 15 min at 16,000 g. The supernatant was boiled in SDS loading buffer. After SDS-PAGE, the samples were transferred to a nitrocellulose membrane. Primary antibodies to the following targets were used: GAPDH (Proteintech, 60004-1-Ig, 1:8000), TH (Sigma-Aldrich, AB152, 1:1000), p-Ser129 α-synuclein (Cell Signaling Technology, 23706 S, 1: 1000), Cofilin 1 (Cell Signaling Technology, 5175 S, 1:1000). The membranes were incubated with primary antibodies overnight at 4 °C. The membranes were washed 3 times in PBST and incubated with HRP-conjugated secondary antibodies. The signals were developed using enhanced chemiluminescent (ECL) substrates. The experiment was repeated at least three times. All blots derived from the same experiment and were processed in parallel.
6
Immunofluorescence Staining of Mouse Brain
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Mice were deeply anesthetized with isoflurane and perfused transcardially with 4% paraformaldehyde in 0.1 M sodium phosphate buffer. Brains were post-fixed for 1–3 days, sectioned coronally (50 μm in thickness) using a vibratome (Leica; VT1000S) and processed for immunofluorescence staining for tyrosine hydroxylase (Millipore; AB152, 1:2000), Gat1 (Cell Signaling; 37342, 1:200), Aldh1a1 (R&D Systems; AF5869, 1:200), HA (Abcam; ab18181, 1:500) or Cre (Synaptic Systems; 257 004, 1:500). After primary antibodies incubation (4°C, overnight), the following secondary antibodies (all from Thermo Fisher Scientific) were applied for 2 h at room temperature: Goat anti-mouse IgG Alexa Fluor 568 (A11004), Goat anti-mouse IgG Alexa Fluor 488 (A11029), Goat anti-rabbit IgG Alexa Fluor 488 (A11034), Goat anti-rabbit IgG Alexa Fluor 568 (A11036), Donkey anti-goat IgG Alexa Fluor 568 (A11057), Donkey anti-rabbit IgG Alexa Fluor 488 (A21206). Brain sections were mounted on Superfrost slides and coverslipped with DAPI Fluoromount-G (SouthernBiotech; 0100-20).
7
Antibody Characterization for Neuronal Markers
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The following primary antibodies were used for the experiments: mouse monoclonal antibodies against β-Tubulin, clone TUB 2.1 (Sigma; T4026; 1:500), anti-Nurr1 (Abcam; ab41917; 1:1000) and anti-GFP (Roche; 11814460001; 1:10,000); rabbit polyclonal antibodies against TH (Millipore; AB152; 1:250), anti-Myc (Cell Signaling; 2272; 1:1000), anti-Pitx3 (Abcam; ab30734; 1:1000), anti-pan-synuclein (Abcam; ab6176; 1:500), anti-γ-synuclein (Abcam; ab6169; 1:1000) and anti-CaMKIIβ (Abcam; ab34703; 1:1000). For immunocytochemistry, secondary antibodies conjugated with Cy2/Cy3 fluorophores (Dianova; 115-225-072/111-165-006; 1:500) were used. Nuclear counterstaining was done with 4′, 6′-diamidino-2-phenylindole (DAPI; 2 µg/ml; Invitrogen; D3571). For Western blot, anti-mouse HRP (Dianova; 715-035-150; 1:5000) or anti-rabbit HRP (Dianova; 711-035-152; 1:5000) secondary antibodies were used. Protein levels were normalized to Actin clone c4 (Millipore; MAB1501).
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