The following primary antibodies were used: ACTB/β-actin (Sigma, A5441; Western blot [WB] 1:100.000); DS-red (Clontech, 632,496; immunocytochemistry [ICC], 1:500); GFP (Abcam, ab13970; ICC 1:5000, WB 1:10.000); HA-epitope-tag (Covance, MMS-101 R; WB 1:1000); LAMP1 (BD Bioscience, 555,798; ICC, 1:3000); LAMP2 (BD Bioscience, 555,803; ICC 1:3000); LC3B (Novus, NB100-2220; WB 1:1000); LC3B (clone EPR18709, Abcam, ab192890; ICC 1:3000); PSEN1 (clone EP2000Y, Abcam, ab76083; WB 1:1000); PSEN2 (clone EP1515Y, Abcam, ab51249; WB 1:1000, ICC 1:3000); RAB5 (Abcam, ab18211; ICC 1:1000); RAB9 (Cell Signaling, 5118; ICC, 1:1000); TFEB (Cell Signaling, 37,785; ICC 1:1000); TFEB (Cell Signaling, 4240; WB 1:1000); VPS35 (Abcam, ab10099; ICC 1:500). Fluorescent Alexa Fluor conjugated (Invitrogen, A11001, A11008, A11039, A21428 or A21422) or horseradish peroxidase (HRP)-conjugated (Dianova, 49,988 or 49,533) secondary antibodies were used.
β actin actb
Manufactured by Merck Group
Sourced in United States
β-actin (ACTB) is a highly conserved cytoskeletal protein that is ubiquitously expressed in all eukaryotic cells. It is a key component of the microfilament system and plays a fundamental role in maintaining the structural integrity of cells, as well as facilitating various cellular processes such as cell motility, cell division, and intracellular transport.
Automatically generated - may contain errors
Lab products found in correlation
Np0007, by Thermo Fisher Scientific (3 mentions)
Imagequant tl, by Cytiva (3 mentions)
Np0005, by Thermo Fisher Scientific (3 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Nxa931, by GE Healthcare (2 mentions)
Fkbp5 fkbp5, by Cell Signaling Technology (2 mentions)
Vcl vinculin, by Merck Group (2 mentions)
Np0002, by Thermo Fisher Scientific (2 mentions)
Tuba4a tuba1a α tubulin, by Merck Group (2 mentions)
Lamp2, by BD (1 mentions)
Lamp1, by BD (1 mentions)
Vps35, by Abcam (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Histone h3, by Cell Signaling Technology (1 mentions)
Rela s536 p, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Hspd1, by Abcam (1 mentions)
Becn1 beclin1, by Santa Cruz Biotechnology (1 mentions)
19 protocols using β actin actb
1
Comprehensive Antibody Panel for Cellular Imaging
2
Western Blot Analysis of Protein Targets
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After PBS rinsed, the cells were lysed in RIPA buffer (1% NP40, 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.25% sodium deoxycholate, 0.1% SDS) plus protease inhibitor cocktail. The protein samples from the cells were separated by a 10% (w/v) SDS-PAGE gel and electrophoretically transferred from the gel to nitrocellulose membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% (w/v) skim milk and then incubated overnight at 4 °C with primary antibodies against HSPD1 (Abcam, MA, USA), E-cadherin, RelA, RelAS536-P, β-catenin, H3 histone, GAPDH (Cell Signaling Technology Inc. Danvers, MA, USA), hemagglutinin (HA) tag (Roche Diagnostics, Rotkreuz, AG, Switzerland) and ACTB (β-actin) (Sigma-Aldrich Corporation, St. Louis, MO, USA). The proteins were probed with horseradish peroxidase-labeled secondary antibody and detected with an ECL reagent. The protein expression level on the membrane was analyzed and measured with the ChemiDoc XRS Imaging System (Bio-Rad) and ImageJ software.
3
Protein Expression Analysis in Cancer Cells
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Western blotting using whole-cell protein extracts and immunohistochemistry (IHC) were performed as previously described.78 (link) Antibodies used were against ERBB2 (Cell Signaling, 2165); Ki67 (Leica Microsystems, NCL-L-Ki67-MM1); LC3B (Novus Biologicals, NB100-2331); BECN1/Beclin1 (Santa Cruz Biotechnology, sc-11427); SQSTM1 (p62, Enzo Life Sciences, BML-PW9860); ATG7 (A2856), ACTB/β-Actin (Sigma-Aldrich, A4527). Cleaved CASP3 IHC was performed by Rutgers Cancer Institute of New Jersey Tissue Analytical Services. Densitometry analysis was performed by ImageJ.79
4
Antibody Dilution Protocol for Protein Analysis
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Antibodies from Abcam were diluted as follows: recombinant anti-PIP5K1C (ab109192) 1:1000, PIP5K1B (ab154818) 1:1000, CHUK/IKK alpha 1:1000 and VCL/vinculin (ab219649) 1:10,000. Antibodies from Cell Signaling Technology were diluted as follows: PIP5K1A (9693) 1:1000 SQSTM1 (8025) 1:5000, LC3A/B (12,741) 1:5,000, p-RPS6 S240/244 (5364) 1:20,000, RPS6 (2217) 1:20,000, and HRP-conjugated secondary anti-rabbit (7074) 1:10,000. Antibodies from Sigma-Aldrich were PIP4K2C (SAB1407977) 1:10,000, ACTB/β-actin (A5441) 1:20,000, and anti-mouse (A4416) 1:10,000. Other antibodies were PIKFYVE (Millipore, MABS522) 1:1000
5
Quantifying Microglial Gene Expression
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RNA was isolated from microglial BV2 cells using Monarch Total RNA Miniprep Kit (New England Biolabs, Massachusetts, USA), and cDNA was generated from 1 μg RNA and random hexamer primers using the SuperScript II Reverse Transcriptase Kit (ThermoFisher, California, USA). qT-PCR was run with the Platinum SYBR Green qPCR SuperMix (ThermoFisher, California, USA) using a Rotor-Gene Q cycler (Qiagen, Hilden, Germany). Primers against mouse CALR, LGALS3, and IL6 were used, with ACTB (β-actin) as the internal control (Sigma, Missouri, USA). Relative mRNA levels of target genes were analyzed by comparing fold-changes in the delta-delta threshold cycle, after normalizing against the internal control for each condition.
6
Autophagy Markers Quantification in Cell Lines
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HeLa and NSC-34 cells were seeded at a density of 2 × 105 cells/well in 12-well plates. Twenty-four h later cells were treated with varying concentrations of BafA1 (0, 1, 10 and 20 nM). Sixteen h after treatment the media was removed from the cells, the cells were washed in PBS and then harvested in fresh PBS. Cells were then pelleted at 380 g for 5 min in a table-top centrifuge and the PBS was removed. Cells were lysed in 300 μL of RIPA buffer (150 mM NaCl, 1% IGEPAL® CA-630 [Sigma-Aldrich®, I8896], 0.5% sodium deoxycholate [Sigma-Aldrich®,D6750], 0.1% SDS [Fisher Scientific, BP166], 50 mM Tris, pH 8.0, including protease [Sigma-Aldrich, P8340] and phosphatase [Sigma-Aldrich, P5726] inhibitors [at a 1:1000 dilution]). A BCA assay (ThermoFisher Scientific, 23225) determined the total protein concentration of each lysate and then lysates were separated by SDS PAGE on a 5–20% acrylamide gels with 20 μg of total protein loaded per well. Gels were transferred overnight and autophagy marker proteins were revealed by western blotting with the mouse anti-SQSTM1 lck ligand antibody, rabbit anti-SQSTM1 antibody (Enzo Life Sciences, BML-PW9860) and rabbit anti-LC3B antibody (MBL International, PM036). The blots were also stained for ACTB/β-actin (Sigma-Aldrich, A1978) to confirm equal protein loading.
7
Protein Expression and Rho Activation Assay
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Protein pellets and electrophoresis were carried out as previously described (Kato et al. 2015) (link). Overnight primary antibody exposure was performed by using antibodies against PARP and CD44, TWIST (Santa Cruz Biotechnology), ZEB2, SOX2, E-cadherin, TAZ, YAP, phospho-YAP (Cell Signaling), OCT4, NANOG, ALDH1A1, and N-cadherin (Abcam), ACTB (β-actin) (Sigma-Aldrich) or TUBB (β-tubulin) (Thermo-Fisher). Peroxidase-conjugated goat anti-mouse/rabbit/goat IgGs were used as secondary antibodies (Bio-Rad). The reaction was developed with chemiluminescence using Western Lightning ECL Pro (Perkin-Elmer) and the signal was detected by Imagequant LAS 500 system (GE Healthcare Bio-Science AB). Densitometry analysis was made using Adobe Photoshop Software Tools and protein levels were normalized against β-actin protein levels.
RhoA pull-down assays were performed using the Rho activation assay kit according to manufacturer's instructions (Cell Biolabs, San Diego, CA, USA).
RhoA pull-down assays were performed using the Rho activation assay kit according to manufacturer's instructions (Cell Biolabs, San Diego, CA, USA).
8
Western Blot Analysis of Protein Samples
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Lysates (8–15 μg) were prepared in 4X sample buffer (Invitrogen, NP0007) with 0.05 M DTT, and boiled for 10 min at 95°C. Lysates were run on a 4–12% Bis-tris gel at 200 V for 50 min in MES running buffer (Invitrogen, NP0002) with antioxidant (Invitrogen, NP0005). Transfer was performed ON, 20 V for 840 min at 4°C, onto a 0.4-μm nitrocellulose membrane. Blocking was done with 5% milk in 1X TBST (Teknova, T9511). After blocking membranes were probed with the following primaries: FKBP5/FKBP5 (1:100; Cell Signaling Technology, 12,210), HTT (Millipore, MAB2166), polyQ 1C2 (1:500; Millipore, MAB1574), VCL/vinculin (1:500; MilliporeSigma, V9131) or ACTB/β-actin (1:1000–2000; MilliporeSigma, A5441). Membranes were incubated with secondary anti-murine horseradish peroxidase (HRP)-coupled antibodies (1:2500; GE Healthcare, NXA931) or anti-rabbit HRP-coupled antibodies (1:2500; GE Healthcare, NA934 at RT for 2 h in 5% milk TBST solution. Protein bands were detected by chemiluminescence (Pierce ECL; Thermo Fisher Scientific, 32,106). ImageQuant TL (v2005, Amersham Biosciences) was used for densitometry analysis.
9
Western Blotting Protocol for Protein Quantification
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Lysates (8–15 μg) were prepared in 4X sample buffer (Invitrogen, NP0007) with 0.05 M DTT, and boiled for 10 min at 95°C. Lysates were run on a 4–12% Bis-tris gel at 200 V for 50 min in MES running buffer (Invitrogen, NP0002) with antioxidant (Invitrogen, NP0005). Transfer was performed ON, 20 V for 840 min at 4°C, onto a 0.4-μm nitrocellulose membrane. Blocking was done with 5% milk in TBST. After blocking membranes were probed with the following primaries: FKBP5/FKBP5 (1:100; Cell Signaling Technology, 12,210), VCL/vinculin (1:500; MilliporeSigma, V9131) or ACTB/β-actin (1:1000–2000; MilliporeSigma, A5441), or TUBA4A/TUBA1A/α-tubulin (1:1000; MilliporeSigma, T6199). Membranes were incubated with secondary anti-murine HRP-coupled antibodies (1:2500; GE Healthcare, NXA931) or anti-rabbit HRP-coupled antibodies (1:2500; GE Healthcare, NA934) at RT for 2 h in 5% milk TBST solution. Protein bands were detected by chemiluminescence (Pierce ECL; Thermo Fisher Scientific, 32,106). ImageQuant TL (v2005, Amersham Biosciences) was used for densitometry analysis.
10
Western Blot for Protein Quantification
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Lysates (15–30 μg) were prepared in 4X sample buffer (Invitrogen, NP0007) with 0.05 M DTT, and boiled for 10 min at 95°C. The R6/2 lysates were run on a 4–12% Bis-tris gel at 200 V for 50 min in MES running buffer (Invitrogen, NP0002) with antioxidant (Invitrogen, NP0005). The zQ175 lysates were separated on 3–8% tris-acetate gel at 200 V for 90 min in tris-acetate running buffer. Transfer was performed ON, 20 V for 840 min at 4°C, onto a 0.4-μm nitrocellulose membrane. Blocking was done with 5% milk in TBST. After blocking membranes were probed with the following primaries: HTT (1:500; MilliporeSigma, 5492), FKBP5/FKBP51 (1:100; Cell Signaling Technology, 12,210), VCL/vinculin (1:500; MilliporeSigma, V9131) or ACTB/β-actin (1:1000–2000; MilliporeSigma, A5441), or TUBA4A/TUBA1A/α-tubulin (1:1000; MilliporeSigma,T6199). Membranes were incubated with secondary anti-murine HRP-coupled antibodies (1:2500; GE Healthcare, NXA931) or anti-rabbit (1:2500;GE Healthcare, NA934) at RT for 2 h or ON at 4°C in 5% milk TBST solution. Protein bands were detected by chemiluminescence (Pierce ECL; Thermo Fisher Scientific, 32,106). ImageQuant TL (v2005, Amersham Biosciences) was used for densitometry analysis.
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