Ps20 antibodies: Mouse monoclonal 1G7 was generated as previously described [6] (link). C-terminal and N-terminal polyclonal abs specific to ps20 were generated by Eurogentech (Southampton, UK) by inoculation of rabbits with peptides AEEAGAPGGPRQPRA and KNVAEPGRGQQKHFQ respectively (Supplementary Fig. 1 ). Purification was by affinity chromatography to the immunising peptide. Where these antibodies are used in western blotting (WB) they are labelled C-term and N-term respectively. Antibody to V5-tag was from Sigma. Antibody to β-actin was from Santa Cruz Biotechnology. HRP-conjugated goat anti-rabbit and rabbit anti-goat used for WB were from thermo-scientific.
Antibody to β actin
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Antibody to β-actin is a laboratory reagent used to detect and quantify the presence of the β-actin protein in biological samples. β-actin is a highly conserved cytoskeletal protein that plays a crucial role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Phospho cdk1 tyr15, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Iblot2, by Thermo Fisher Scientific (1 mentions)
Cleaved parp asp214, by Cell Signaling Technology (1 mentions)
Complete mini protease inhibitor, by Roche (1 mentions)
Phospho rb ser780, by Cell Signaling Technology (1 mentions)
Cdk4 sc 260, by Santa Cruz Biotechnology (1 mentions)
Pten 9188s, by Cell Signaling Technology (1 mentions)
Chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Fluorescently labeled secondary antibody, by LI COR (1 mentions)
Anti nanog antibody, by Santa Cruz Biotechnology (1 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Ab59337, by Abcam (1 mentions)
Human tnf α, by R&D Systems (1 mentions)
Ab16502, by Abcam (1 mentions)
Phospho eif2α, by Cell Signaling Technology (1 mentions)
Eif2α, by Cell Signaling Technology (1 mentions)
12 protocols using antibody to β actin
1
Monoclonal and Polyclonal Antibodies for Ps20 Protein
2
Protein Extraction and Western Blot Analysis
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Cells were washed once with cold PBS and upon removal, cells were lysed in radio-immunoprecipitation assay (RIPA) buffer supplemented with PhosSTOP phosphatase and CompleteMini protease inhibitors (Roche, Switzerland) for protein extraction. Proteins were separated by polyacrylamide gel electrophoresis PAGE using 4-12% gradient gels followed by protein transfer onto nitrocellulose membranes with iBLOT2 (ThermoFisher Scientific, Waltham, MA). Membranes were then blocked in 5% nonfat dry milk in Tris- buffered saline with Tween-20 (TBST) and incubated at 4oC with primary antibodies. Proteins of interest were detected with horseradish peroxidase-conjugated secondary antibodies and Advansta WesternBright™ ECL Spray was used for detection (Thomas Scientific, Swedesboro, NJ). The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): phospho-RB (Ser780) (#3590), RB (#9309), Cyclin D1 (#2978), phospho-CDK1 (Tyr15) (#4539), CDK1 (#77055), Wee1 (#13084), gamma-H2AX (Ser139) (#80312), H2aX (#7631) and cleaved PARP (Asp214) (#5625). Antibody to p53 (#ab32389) was purchased from Abcam (Cambridge, UK). For loading control, antibody to β-actin (#sc-47778) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
3
Western Blot Analysis of Signaling Proteins
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The total proteins were extracted with lysis buffer solution supplemented with protease inhibitors and phosphatase inhibitors (Pierce, USA) and quantified by the BCA protein assay (Pierce, USA). Each sample (30 μg) was separated by 10% SDS-PAGE, and then transferred to nitrocellulose (NC) membranes (PALL, USA). After blocking with 5% BSA in Tris-buffered saline-Tween 20, the membrane was incubated with primary antibody for overnight at 4°C. PHRF1 (ab85974) was purchased from Abcam. Akt (sc-8312), p-Akt (sc-33437), TGIF (sc-9084), p38 (sc-535), p-p38 (sc-17852-R), p21 (sc-397), cyclin A (sc-751), cyclin B1 (sc-752), cyclin D1 (sc-718) and CDK4 (sc-260) were purchased from Santa Cruz Technology. Rb (#9313S), p-Rb (#8516S), c-Myc (#13987S), p65 (#8242S) and PTEN (#9188S) were purchased from Cell Signaling Technology. After incubation with peroxidase-coupled anti-rabbit-IgG (ZSGB-BIO, Beijing, China) at room temperature for 1h, the protein bands were visualized by Bio-Rad Clarity™ western ECL substrate (Bio-Rad, USA) in the ChemiDoc™ XRS+ Imaging System (Bio-Rad, USA). Antibody to β-actin (1:1000, Santa Cruz Technology, sc-8432) was used as a loading control.
4
Western Blot Analysis of Stem Cell Markers
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Whole-cell lysates were dissolved in lysis buffer [150 mmol/L NaCl, 5 mmol/L EDTA, 1% Triton X-100, and 10 mmol/L Tris (pH 7.4)] with protease inhibitor (Roche; Mannheim, Germany) and subjected to electrophoresis on 4% to 15% Tris-glycine gels using 50–80 µg of protein per lane. Protein levels were first determined by the DC protein Assay (Bio-Rad; Hercules, CA). A Bio-Rad Trans-Blot system was used to transfer the proteins to Immobilon-P FL membranes (Millipore; Bedford, MA). Blots were incubated overnight at 4°C in 5% dry nonfat milk in TBS (150 mmol/L NaCl, 300 mmol/L KCl, 10 mmol/L Tris (pH 7.4), 0.01% Tween 20). Blots were incubated for 16 h at 4°C with anti-POU5F1 antibody at a dilution of 1∶500 (Santa Cruz; Santa Cruz, CA), anti-NANOG antibody at a 1∶100 dilution (Santa Cruz; Santa Cruz, CA), or antibody to β-actin (Santa Cruz; Santa Cruz, CA). A fluorescently labeled secondary antibody (Li-Cor; Lincoln, NE) was dissolved in 5% milk in the TBS-T buffer and incubated with the blot for 2 h at room temperature. After three 10 min washes, blots probed with fluorescently labeled antibody were imaged using an Odyssey Infrared Imager (Li-Cor; Lincoln, NE).
5
Investigating Transcription Factor Regulation
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Antibodies to Kaiso (6F/6F8), Glucocorticoid Receptor (BuGR2), phosphor-p65 (ab86299), p65 (ab16502), and Sgk-1 (ab59337) were purchased from Abcam (Cambridge, MA, USA). Antibody to β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human TNF-α was purchased from R&D Systems (MN, USA). Other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
6
Investigating Molecular Pathways in LPS-Induced Stress
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Lipopolysaccharide (LPS) from Escherichia coli O55:B5 was obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies to β-catenin, caspase 3, cleaved caspase 3, PARP [poly(ADP-ribose) polymerase], cleaved PARP, phosphor-PERK (protein kinase R-like endoplasmic reticulum kinase), CHOP (CCAAT-enhancer-binding protein homologous protein), eIF-2α (eukaryotic translation initiation factor), phospho-eIF-2α, CREB, and anti-rabbit IgG antibody as a second antibody were purchased from Cell Signaling Technology (Beverly, MA, USA). Wnt agonist was obtained from Calbiochem (Cat. No. 681665; San Diego, CA, USA). The antibody to β-actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Caspase inhibitor (Z-VAD-FMK) and caspase 3 inhibitor (DEVD-FMK and IETD-FMK) were obtained from MBL (Nagoya, Japan).
The appropriate concentrations of inhibitors and antibodies were determined in preliminary experiments.
The appropriate concentrations of inhibitors and antibodies were determined in preliminary experiments.
7
Comparative Analysis of Cancer Cell Lines
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Human lung cancer cells, LNM35 and A549, were maintained in RPMI 1640 (Hyclone, Cramlington, UK), human breast cancer cells, MDA-MB-231 and T47D; human colon cancer cells HT-29, HCT-116, and HCT8/S11; and mouse gastric stem cells (MGSC) were maintained in DMEM (Hyclone, Cramlington, UK). All media were supplemented with antibiotics (penicillin, 50 U/ml; streptomycin, 50 µg/ml) (Hyclone, Cramlington, UK) and with 10% foetal bovine serum (Hyclone, Cramlington, UK). In all experiments, cell viability was higher than 99% using trypan blue dye exclusion. The culture medium was changed every 3 days, and cells were passaged once a week when the culture reached 95% confluence. PTC-209 was purchased from Xcess Biosciences Inc (Xcess Biosciences Inc, San Diego, CA). Frondoside A, camptothecin, cisplatin, oxaliplatin, and 5-fluorouracil were purchased from Sigma-Aldrich (Saint Louis, MO). Antibodies to β-tubulin, gp130, STAT3, phospho-STAT3 were obtained from Cell Signaling Technology (Cell Signaling, Beverly, MA). Bmi-1 antibody was obtained from Abcam (Cambridge, UK). Antibody to β-actin was obtained from Santa Cruz Biotechnology, Inc (Santa Cruz, CA).
8
Protein Signaling Pathway Analysis
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Antibodies to pSTAT3 (tyrosine 705), pJAK3, JAK3, and PTPN6 were purchased from Cell Signaling Technologies (Beverly, MA, USA) Antibody to β-Actin was purchased from Santa Cruz (Dallas, TX, USA). P-tyrosine (4G10) antibody was purchased from Millipore. The pharmacological inhibitors romidepsin and azacytidine were purchased from Sigma-Aldrich. JAK3 inhibitor WHIP-154 was purchased from Santa Cruz Biotechnology and JAK2 inhibitor ruxolitinib was procured from ChemieTek (Indianapolis, IN).
9
Quantitative Western Blot Protocol
10
Protein Expression Analysis of Breast Cancer Cell Lines
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Total proteins were extracted from breast cancer cell lines (MDA-MB-453, MDA-MB-231, MCF-7 and SK-BR-3) using RIPA Lysis Buffer (Beyotime, Jiangsu, China) and separated by SDS-PAGE (10%) (Beyotime, Jiangsu, China). These proteins were transferred to 0.45 μm polyvinylidene difluoride membrane (Millipore, Massachusetts, USA) by Bio-Rad transference chamber (Bio-Rad Laboratories, California, USA). Primary antibody to ILT4 (R & D, Minneapolis, USA; dilution 1:500) was used to detect the protein expression. The membrane was incubated with appropriate secondary antibodies labeled to horseradish peroxidase (Santa cruz, California, USA; dilution 1:5000). The protein levels were normalized by reprobing the blots with antibody to β-actin (Santa cruz, California, USA; dilution 1:1000). The signals were detected by the ECL Plus Western Detection Kit (Beyotime, Jiangsu, China) and recorded on the Kodak X-ray films. The protein expression was determined utilizing the BandScan 5.0 software.
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