Protein extracts from all control, NC, and experimental groups of cells were prepared using RIPA lysis buffer and protein concentrations were determined using the BCA Protein Assay kit (both Beyotime Institute of Biotechnology; Haimen, China). Samples (40 µg) were separated using SDS-PAGE on 10% gels and transferred onto polyvinylidene fluoride membranes. After incubation in 5% skim milk for 2 h at room temperature to block non-specific binding, the membrane was incubated for 16 h at 4°C with primary antibodies (all purchased from Abcam, Cambridge, UK; dilution, 1:500) against KLF7 (#ab197690), E-cadherin (#ab15148), N-cadherin (#ab76057), vimentin (#ab137321), β-actin (#ab16039) and snail (#ab82846) diluted in PBS containing 3% BSA. The membrane was then washed 3 times with TBST (20 mM Tris-HCl, 137 mM NaCl and 0.1% Tween-20; pH 7.6) for 10 min/time, and incubated with anti-rabbit IgG-HRP antibodies (SC-2370, 1:5,000; Santa Cruz Biotechnology, Inc., Dallas, TX). Finally, the membrane was washed with TBST for 10 min 3 times. Following a further wash with TBST, signals were visualized using a Chemiscope5600 (Shanghai Clinx Science Instruments Co., Ltd., Shanghai, China) using MaxiLumin chemiluminescence detection reagents (Biokit, Barcelona, Spain).
Ab16039
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab16039 is an antibody that can be used for laboratory research purposes. It is a primary antibody that binds to a specific target. The core function of this product is to serve as a research tool for detecting and studying the target of interest. No further details can be provided without extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (3 mentions)
Protein assay kit, by GE Healthcare (2 mentions)
Medical x ray film, by GE Healthcare (2 mentions)
Pre stained protein ladder 10 250kda, by GE Healthcare (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Verapamil, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ab15148, by Abcam (1 mentions)
Ab137321, by Abcam (1 mentions)
Ab82846, by Abcam (1 mentions)
Ab76057, by Abcam (1 mentions)
Ab197690, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ecl plus kit, by GE Healthcare (1 mentions)
Ab113, by Abcam (1 mentions)
Ecl system rpn2232, by Cytiva (1 mentions)
Hatf00010, by Merck Group (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Easypack protease inhibitors, by Roche (1 mentions)
12 protocols using ab16039
1
Western Blot Analysis of EMT Markers
2
Western Blot Analysis of PPM1F in Cell Lines
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HepG2 and LO2 cell lines were harvested and extracted using lysis buffer. Cell extracts were boiled in loading buffer and equal amount of cell extracts were separated on 15% SDS-PAGE gels. Separated protein bands were transferred into polyvinylidene fluoride (PVDF) membranes. The primary antibodies against PPM1F (ab156222, Rabbit polyclonal antibody, Abcam, Cambridge, MA, USA) and β-actin (ab16039, Rabbit polyclonal antibody, Abcam, Cambridge, MA, USA) were diluted at a ratio of 1:1000 according to the instructions and incubated overnight at 4 °C. Horseradish peroxidase-linked secondary antibodies were added at a dilution ratio of 1:10000, and incubated at room temperature for 1 h. The membranes were washed with PBS for three times and the immunoreactive bands were visualized using ECL-PLUS/Kit (GE Healthcare, Piscataway, NJ, USA) according to the kit’s instruction.
3
Western Blot Analysis of Tyrosine Hydroxylase
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Proteins were extracted from SN tissues with lysis buffer (0.1 M Tris at pH 7.4, 100 × Na-orthovanadate, pH 7.4) supplemented with a protease inhibitor cocktail (P2714; Sigma-Aldrich). The same amount of proteins from each sample were separated on a 10% SDS-PAGE gel, transferred to a nitrocellulose membrane (HATF00010; Millipore) at 4 °C overnight blotting, and hybridized with the primary antibodies tyrosine hydroxylase (TH) (1:1000 dilution; AB113, Abcam, Cambridge, MA, USA) and β-actin (1:1000 dilution; ab16039, Abcam, Cambridge, MA, USA). The membranes were then incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies. According to the manufacturer’s instructions, an ECL system (RPN2232; Amersham Biosciences, Buckinghamshire, United Kingdom) was used to detect antibody-bound proteins, which were then analyzed using ImageJ, 1.37v software.
4
Western Blot Analysis of MBL2 Protein
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LO2 and HepG2 cell lines were harvested and extracted using lysis buffer. Cell extracts were boiled in loading buffer and equal amounts of cell extracts were separated on 15% sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS-PAGE) gels. Separated protein bands were transferred into polyvinylidene fluoride membranes. The primary antibodies against MBL2 (24207-1-AP, rabbit polyclonal antibody; Proteintech, Rosemont, Illinois) and β-actin (ab16039, rabbit polyclonal antibody; Abcam, Cambridge, Massachusetts) were diluted at a ratio of 1:1000 according to the instructions and incubated overnight at 4°C. The specific details were described as previously reported.11 (link)
5
Western Blot Analysis of ChSy-2 Protein
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Protein extracts were prepared from cells using M-PER™ Mammalian Protein Extraction Reagent (cat. no. 78503; Thermo Fisher Scientific, Inc.) supplemented with EDTA-free complete protease inhibitor cocktail (cat. No. 88265; Thermo Fisher Scientific, Inc.). Protein content was determined using a Micro BCA™ Protein Assay Kit (cat. no. 23235; Thermo Fisher Scientific, Inc.). Protein samples (30 μg) were separated on a 12% SDS-PAGE gel and electrophoretically transferred to PVDF membranes (cat. no. 162-017; Bio-Rad Laboratories, Inc., Hercules, CA, USA). After blockage, the membranes were successively incubated with anti-human ChSy-2 or β-actin rabbit antibodies (dilution, 1:1000, ab75052 or ab16039, respectively; Abcam) and goat anti-rabbit IgG antibody conjugated to horseradish peroxidase (dilution, 1:5000; cat. no. ab97051; Abcam). Protein bands were visualized by enhanced chemiluminescence using SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Inc.) on a ChemiDoc™ XRS+ system (Bio-Rad Laboratories, Inc.). Target blots were normalized to β-actin and semiquantified by densitometry using ImageJ software (v2.1.4.7; National Institutes of Health, Bethesda, MD, USA).
6
Quantitative Western Blot Analysis of αSMA
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Tissue samples (50 mg) were solubilized in Ripa Buffer (89900, Thermo Fisher) with the addition of EASYpack protease inhibitors (5892970001, Roche, Switzerland). Protein concentration in supernatants after 14,000 rpm centrifugation was determined using the BCA method. 25 μg of protein were separated on 8% SDS-PAGE with subsequent blotting on a nitrocellulose membrane (BIO-RAD, USA) using Trans-Blot® Turbo™ Transfer System (Bio-Rad, USA) with subsequent immersion in 5% skimmed milk in TBST buffer. Primary ab: anti-αSMA (ab5694, Abcam, UK); anti-beta Actin (ab16039, Abcam, UK). Secondary ab: 1:10,000-anti-Mouse IgG (H + L) secondary antibody (115-005-003, Jackson ImmunoResearch, USA), anti-Rabbit IgG (H + L) secondary antibody (111-035-144, Jackson ImmunoResearch, USA). Signal was revealed by the ECL method following the HRP reaction.
7
Western Blot Analysis of HIF Proteins
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Aliquots of liver homogenate (30 µg) were separated by sodium dodecyl sulfate (SDS) polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. Nonspecific binding was blocked with 5% nonfat milk for one hour and incubated overnight at 4˚C with primary antibodies: HIF-1α antibodies (NB100-479; Novus Biologicals), HIF-2α antibodies (NB100-102; Novus Biologicals) and anti-β-actin antibodies (ab16039; Abcam). Membranes were washed with PBS with Tween-20 (PBST) three times for 10 min and then incubated with a secondary goat anti-rabbit antibodies (1:5,000) for one hour at 37˚C. Finally, the membranes were washed with PBST three times for 10 min and developed with the ECL system (GE Healthcare).
8
Immunoblotting of TRPV2 Protein
9
Evaluating P-glycoprotein Expression
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Clemastine fumarate, digoxin, verapamil, Rho123, 3-(4,5-dime-thylthiazol-2-thiazolyl)-2,5-diphenyl-tetrazoliumbromide (MTT), penicillin-streptomycin, and protease inhibitor cocktail were obtained from Sigma–Aldrich (St. Louis, MO, USA). Anti-P-gp mouse monoclonal antibody (ab80594) and rabbit polyclonal antibody to beta Actin (ab16039) were purchased from Abcam (USA). Horseradish peroxidase (HRP) conjugated anti-mouse immunoglobulin (IgG) (AP8036) and anti-rabbit IgG (AP7181) were purchased from Razi Biotech (Iran). Dulbecco’s modified Eagle medium (DMEM), trypsin-EDTA (0.25%), fetal calf serum (FCS) were purchased from Gibco (Carlsbad, CA, USA). Tissue culture flasks and other disposable cell culture items were purchased from TPP Co. (Switzerland). Enhanced chemiluminescence (ECL) Western blot detection kit, medical X-ray film, pre-stained protein ladder (10-250kDa), and protein assay kit were purchased from Amersham (GE Healthcare, Chalfont St. Giles, UK)), Fuji (Tokyo, Japan), Cinagen, and Pars Azmoon (Iran), respectively. All other chemicals were purchased from Merck Co. (Darmstadt, Germany).
10
Western Blot Analysis of Cellular Signaling
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The following antibodies and reagents were used for this study: rabbit anti-p53 (1:500, ab1431; Abcam), rabbit anti-p53 (acetyl K382) (1:1,000, ab75754; Abcam), rabbit anti-p21 (1:500, ab109520; Abcam), rabbit anti-gamma H2AX (phospho S139) (1:1,000, ab2893; Abcam), rabbit anti-ERK1/2 (1:500, No 5013; Cell Signaling Technology Inc., Danvers, MA, USA), rabbit anti-phospho-ERK1/2 (Thr202/Tyr204) (1:2,000, No 4376; Cell Signaling Technology Inc.), rabbit anti-PKM2 (1:1,000, ab150377; Abcam), rabbit anti-β-actin (1:2,000, ab16039; Abcam), mouse anti-Tubulin (1:2,000, AT819; Beyotime Institute of Biotechnology), rabbit anti-active caspase3 (1:1,000, ab49822; Abcam), mouse anti-cleaved PARP1 (1:1,000, ab198490; Abcam), mouse anti-cytochrome C (1:2,000, ab13575; Abcam), rabbit anti-VDAC (1:1,000, ab15895; Abcam), mouse anti-Bcl-2 (1:1,000, ab201566; Abcam), rabbit anti-Bax (1:500, No 2774; Cell Signaling Technology Inc.), and mouse-anti-HA (1:500, H3663; Sigma-Aldrich Co.). Res-veratrol (Cat. No: R5010) was purchased from Sigma-Aldrich Co. TEPP-46 (HY18657-4) was purchased from MCE (Med-ChemExpress Inc., Monmouth Junction, NJ, USA).
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