To evaluate the specificity of PTEN-antibody used for the immunohistochemical study, cell lysates were prepared and Western blot analysis was performed. The primary antibodies for PTEN, ATR, and β-actin used in this study were incubated at room temperature for 1 h (PTEN 1:100 dilution [Dako], ATR 1:1000 dilution [Cell signaling] and β-actin 1:5000 dilution [Sigma]). Infrared dye-labelled secondary antibodies (Li-Cor) [IRDye 800CW Donkey Anti-Rabbit IgG and IRDye 680CW Donkey Anti-Mouse IgG] were incubated at a dilution of 1:10,000 for 1 h. Membranes were scanned with a Li-Cor Odyssey machine (700 and 800 nm) to determine protein expression.
β actin
Manufactured by Agilent Technologies
Sourced in Denmark, China
β-actin is a structural protein that is ubiquitously expressed in eukaryotic cells. It is a key component of the cytoskeleton and plays a critical role in cellular processes such as cell motility, cell division, and cell signaling.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (1 mentions)
Stat3, by Agilent Technologies (1 mentions)
Pstat3, by Agilent Technologies (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
P p38α, by Cell Signaling Technology (1 mentions)
P erk, by Agilent Technologies (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Pierce ecl substrate western blot detection system, by Thermo Fisher Scientific (1 mentions)
Anti pcna, by Cell Signaling Technology (1 mentions)
Molecular imager chemidoc xrs system, by Bio-Rad (1 mentions)
6 protocols using β actin
1
Validating PTEN Antibody Specificity
2
Immunoblotting of LCL Samples
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3
MPTP-induced Neuroinflammation Protein Analysis
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Striata was dissected immediately after sacrificing miceon seventh day of MPTP-induction (n = 6). The tissue samples were thawed and homogenized with lysis buffer, then centrifuged to collect the supernatant and stored at-80 °C until the day of western blot assay. Total protein (40 µg) from each sample was heated with sodium dodecyl sulphate (SDS) sample buffer at 70 °C for 60 s and the proteins were separated on SDS polyacrylamide gels (10%) and transferred to nitrocellulose membranes by electrophoresis. Primary mouse antibodies for TNF-α, iNOS, IL1β, NFkB1, β-actin (1:1200, Dako) were incubated with the membranes followed by horse anti-rabbit secondary antibody (1:400). The differences in the band density of proteins were detected using ECL kit and measurements were made using ChemiDoc system (Biorad). Relative protein expression levels with respect to β-actin protein levels were calculated.
4
Molecular Signaling Pathway Analysis
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All the chemicals used in this study were obtained from Invitrogen, Carlsbad, USA. The β-actin, AKT, and p38α were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The p-AKT, p-p38α, and p-ERK were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit polyclonal antibodies against p38α, p-p38α, p-Akt, and p-ERK were purchased from DAKO (Glostrup, Denmark). Goat polyclonal antibodies against Akt and β-actin were purchased from DAKO.
5
Monoclonal Antibodies for Immunohistochemistry
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Mouse monoclonal antibodies to CD8 (C8/144), CD45RA (4KB5), and CD68 (KP1) were produced in the authors’ laboratories. Antibodies to CD20 (Clone L26), cytokeratins (Clones Lp34 and Clones MNF116), and β-actin (diluted 1:100) were obtained from DakoCytomation (Glostrup, Denmark).
6
Protein Extraction and Western Blot
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Total proteins were extracted using CHAPS lysis buffer. Proteins were separated by sodiem dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membrane. Blots were blocked in 4% dry milk at room temperature for 1 h, and immuno-stained with primary antibodies (anti-PCNA, 1:2,000; Cell signaling Technology, Shanghai, China and β-actin 1:10,000; Dako, Glostrup, Denmark) at 4°C overnight. The results were visualized via a chemiluminescent detection system (Pierce ECL Substrate Western-blot detection system; Thermo, Rockford, IL) and exposed in Molecular Imager ChemiDoc XRS System (Bio-Rad, Hercules, CA).
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