Western blots were carried out as described previously (Ventoso et al., 2006 (link)) using the following primary antibodies: anti-RPS4X (sc-85133, Santa Cruz Biotech.), anti-RPS7 (sc-377317, Santa Cruz Biotech.), anti-RPS6 (sc-74576, Santa Cruz Biotech.), anti-eIF4A (STJ2724, St. John´s lab,), anti-eIF3g (STJ23512, St. John´s lab), anti-EGFP (11814460001, Roche), anti-eEF1A1 (2551, Cell Signaling), anti-AKT1 (9272, Cell Signaling), anti-ACTB (T-5168, Sigma), anti-eIF4b (sc-376062, Santa Cruz Biotech.), anti-HRas (sc-53959, Santa Cruz Biotech.), anti-CCND3 (sc-453, Santa Cruz Biotech.), anti-ODC1 (sc-398116, Santa Cruz Biotech.), anti-GRK2 (a gift from C. Murga, CBMSO). Blots were developed with ECL (GE) and bands were quantified by densitometry. Immunofluorescence analysis was carried out as described previously using anti-RPS7 (1:500) and anti-mouse Alexa 595 as secondary antibody (Invitrogen). The preparations were analyzed under a Nikon A1R confocal microscope.
Anti actb
Manufactured by Merck Group
Sourced in United States
Anti-ACTB is a laboratory reagent that specifically binds to the beta-actin (ACTB) protein. It is used in various research applications to detect and quantify the expression of ACTB, a commonly used reference gene or loading control in molecular biology experiments.
Automatically generated - may contain errors
Lab products found in correlation
Anti flag, by Merck Group (4 mentions)
Anti lc3b, by Merck Group (4 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti src, by Cell Signaling Technology (2 mentions)
Biospectrum imaging system, by Analytik Jena (2 mentions)
Anti gapdh, by Merck Group (2 mentions)
Anti akt1, by Cell Signaling Technology (1 mentions)
Anti rps6, by Santa Cruz Biotechnology (1 mentions)
Anti egfp, by Roche (1 mentions)
Anti hras, by Santa Cruz Biotechnology (1 mentions)
Anti ccnd3, by Santa Cruz Biotechnology (1 mentions)
Anti eef1a1, by Cell Signaling Technology (1 mentions)
Anti eif4b, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Nb300 318, by Novus Biologicals (1 mentions)
Nb110 59 906, by Novus Biologicals (1 mentions)
Alexa fluor 594 donkey anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
3 methyladenine m9281, by Merck Group (1 mentions)
Anti lc3b nb100 2220, by Novus Biologicals (1 mentions)
40 protocols using anti actb
1
Western Blotting and Immunofluorescence Analysis
2
Immunofluorescence and Immunoblot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
5000 cells were seeded in laminin-coated 24-well plates. After fixation with 4% paraformaldehyde, cells were permeabilized with 0.1% Triton X100 for 15 min at room temperature. The cells were then incubated overnight at 4 ℃ with CIC antibody (NB110-59,906, NOVUS). Samples were rinsed three times in PBS and incubated for 1 h at room temperature with AlexaFluor 594 donkey anti-rabbit secondary antibody (Thermo Fisher Scientific). Nuclei were stained with 0.5 µg/ml DAPI for 1 min at room temperature. Images were acquired using fluorescence microscopy. The primary antibodies used for the immunoblots: anti-CIC (NB110-59,906, NOVUS), anti-ETV4 (10,684–1-AP, Proteintech), anti-xCT (NB300-318, NOVUS), anti-ACTB (A3854, Sigma), anti-GAPDH (2118, Cell Signaling).
3
Molecular Mechanisms of Mitophagy Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The immortalized RPTC cell line was originally obtained from Dr. Ulrich Hopfer (Case Western Reserve University) and maintained in DMEM/F-12 medium supplemented with 10% fetal bovine serum and growth factors [47 ]. The retrovirus packaging cell line (293-Phoenix) was provided by Drs. Xiongjie Jin and Nahid F. Mivechi (Augusta University). The cells were cultured in DMEM medium with 10% fetal bovine serum. Primary antibodies: anti-LC3B (NB100-2220), anti-PINK1 (BC100-494) and anti-FUNDC1 (NBP1-81063) were from Novus Biologicals; anti-SQSTM1 (ab56416), anti-IMMT/MIC60 (ab110329), anti-COX4I1 (ab153709) and anti-PPIB (ab16045) were from Abcam; anti-TOMM20 (sc-11415), anti-PINK1 (sc-517353), anti-PRKN (sc-133167), anti-BNIP3L/NIX (sc-166332) and anti-HSPD1 (sc-13966) were from Santa Cruz Biotechnology; anti-CASP3 (9665) and anti-cleaved CASP3 (9664) were from Cell Signaling Technology; anti DNM1L (BD Biosciences, 611113); anti-ACTB (Sigma-Aldrich, A5316). Secondary antibodies for immunoblot analysis were from Jackson ImmunoResearch Laboratories. CCCP (C2759), chloroquine (C6628) and 3-methyladenine (M9281) were from Sigma-Aldrich. BECN1 peptide (Tat-BECN1, sequence: YGRKKRRQRRRGGTNVFNATFEIWHDGEFGT) and its control peptide (Tat-scrambled, sequence: YGRKKRRQRRRGGVGNDFFINHETTGFATEW) were synthesized by GenScript.
4
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were briefly rinsed in PBS and lysed with RIPA buffer (1% NP40, 50 mM Tris Cl pH 7.5, 150 mM NaCl, 0.25% sodium deoxycholate 1% SDS, and a protease inhibitor cocktail). The cell lysates were resolved by SDS-PAGE and transferred electrophoretically onto nitrocellulose membranes. The membranes were blocked with bovine serum albumin (BSA) for the anti-phosphoserine antibody and with 0.1% casein in 0.2X PBS for the other antibodies. The membranes were then incubated with the primary antibodies, including anti-Src (2109, cell signaling), anti-pSrc (6943, Cell Signaling), phospho-Stat-3 (9145, cell signaling), and anti-ACTB (A5441, Sigma) at 4°C overnight. The proteins were probed with HRP-labeled secondary antibody and detected with an ECL reagent. The membrane was scanned and analyzed for protein expression with a BioSpectrum® Imaging System (UVP, Upland, CA, United States).
5
Western Blot Analysis of FOXC2, PARP, and cPARP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was prepared from GC cells or tissues using RIPA buffer (Beyotime, Shanghai, China) with a complete protease inhibitor cocktail (Roche, Manheim, Germany). Approximately 20–50 µg of denatured protein was fractionated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked and then incubated with anti-FOXC2 (Abcam, ab5060), anti-PARP (Cell Signaling Technology (CST), #9532), anti-cPARP (CST, #9541), and anti-ACTB (Sigma-Aldrich, A1978) antibodies overnight at 4°C. Proteins were visualized using Dura SuperSignal Substrate (Pierce, USA), and the blots were scanned using a Molecular Imager ChemiDox XRS+ Imaging System with Image Lab software (Bio-Rad Laboratories).
6
Western Blot Analysis of Cell Adhesion Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cell lysates were prepared using NP-40 lysis buffer. Protein concentrations were determined using the Bradford Assay (Thermo Fisher Scientific). Twenty milligrams of each protein lysate were loaded onto 4–20% precast polyacrylamide gels (Bio-Rad, Hercules, CA, USA), separated by electrophoresis, and electrotransferred to Sequi-Blot PVDF Membranes (Bio-Rad). Primary antibodies for Western blotting were anti-HOXB13 (Cat# PA5-78327; Thermo Fisher Scientific), anti-ITGAV (Cat# PA5-86575; Thermo Fisher Scientific), anti-ITGB5 (Cat# PA5-17260; Thermo Fisher Scientific), anti-ITGB1 (Cat# ab179471; Abcam, Cambridge, MA, USA) anti-ITGB3 (Cat# ab179473; Abcam), anti-ACTB (Cat# A5316; Sigma-Aldrich), anti-CCL2, clone 2D8 (Cat# MABN712; Merck Millipore, Burlington, MA, USA), and anti-IBSP (Cat# 5468; Cell Signaling Technology). Primary antibodies were detected by using appropriate horseradish peroxidase-conjugated secondary antibodies. Signals were then detected with an Enhanced Chemiluminescence System (GE Healthcare, Piscataway, NJ, USA) and blots were scanned using the Image Reader LAS-4000 (Fuji Film, Tokyo, Japan). Signal intensity was quantified using ImageJ software (RRID: SCR_003070; National Institutes of Health, Bethesda, MD, USA).
7
Western Blot Analysis of Protein Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cell lysates were prepared by treating a confluent 10 cm plate of cells with 2x Laemmli sample buffer supplemented with 5% β-mercaptoethanol and 4% sodium dodecyl sulfate. The cell solution was briefly sonicated using a probe sonicator, boiled at 95 °C for 3 min and resolved by denaturing polyacrylamide gel electrophoresis. The proteins were then transferred to a PVDF membrane using a Trans-Blot® Turbo™ Transfer system (Bio-Rad). Membranes were blocked for 1 h at room temperature by incubating with blocking buffer containing 3% bovine serum albumin (BSA) in 1X Tris buffered saline and subsequently incubated at 4 °C overnight with anti-ActB (Sigma Aldrich #A2228) and anti-FLAG (Sigma-Aldrich #F1804) primary antibodies diluted at 1:20,000 and 1:1000 respectively each in blocking buffer. Following overnight incubation, the membranes were washed and incubated for 1 h at room temperature with horse radish peroxidase-conjugated anti-mouse secondary antibody diluted 1:3000 in 5% non-fat dry milk in 1X Tris buffered saline. Immunoreactive bands were detected using SuperSignal™ West Femto Maximum Sensitivity Substrate and a ChemiDoc MP Imaging System (Bio-Rad).
8
Antibody Validation for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-ATG5 (Abcam, ab108327; 1:1000), anti-LC3 (Nanotools, Clone 5F10; 1:1000), anti-ACTB (Sigma, A5441; 1:10,000), anti-ACTIN (Santa Cruz Biotechnology, I-19; 1:5000 [no longer commercially available]), anti-ACTA2/α-SMA (Abcam, ab5694; 1:1000), anti-COL1A1 (Abcam, ab34710; 1:2000), anti-poly UBIQUITIN (Enzo, Clone FK1; 1:5000), anti-turboGFP (Pierce, PA5-22,688; 1:2000), anti-rabbit HRP and anti-mouse HRP (GE Healthcare, NA934V and NA931V; 1:5000)
9
Blue Light Regulation of Tight Junction Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Confluent cells were conditioned in a blue light exposure chamber at indicative illuminances or time intervals. Next, the protein expression level was detected by western blotting, using a previously described protocol [22 (link)]. The antibodies used in this study were as follows: anti-CLDN5 (Abcam, Cambridge, UK), anti-ADAM17, anti-ACTB (Sigma-Aldrich, Chicago, IL), anti-ADAM17 (phospho T735) (Cusabio, Houston, TX), anti-GNAZ (Genetex, Irvine, CA), anti-OCLN (Proteintech, Chicago, IL), and anti-TJP1 (Invitrogen, Waltham, MA, USA).
10
Protein and Histone Mark Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Nuclear extracts were prepared using the Nuclear Extract kit (Active Motif) to examine protein levels of WHSC1L1, and cytoplasmic extracts using the NE-PER nuclear and cytoplasmic extraction kit (78833, ThermoFisher Scientific) were obtained to examine protein levels of cytoplasmic CDK2, CDC6 and ACTB. Primary antibodies used were anti-WHSC1L1 (11345-1-AP, Proteintech, dilution: 1:5000), anti-CDK2 (#2546, Cell Signaling Technology, Danvers, MA, dilution: 1:1000), anti-CDC6 (#3387, Cell Signaling Technology, Danvers, MA, 1:1000), anti-ACTB (A5441, Sigma-Aldrich, St. Louis, MO, dilution: 1:5000) and anti-HA (H6908, Sigma-Aldrich, St. Louis, MO, dilution 1:2000). For detection of histone marks, nuclear extracts were prepared using the Nuclear Extract kit (Active Motif) and 2.5 μg of each extract was loaded for each experiment. Antibodies used were anti-H3K36me2 (07-369, Merck Millipore, dilution: 1:10000), and anti-H3 (ab1791, Abcam, Cambridge, UK, dilution: 1:50000).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!