Lysate proteins (20 μg) from treated cells were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a NC filter. This filter was then immunoblotted with the antibodies of LC3 (L7543, Sigma–Aldrich, 2000-fold diluted), Atg5 (2630, Cell Signaling Technology, Danvers, MA, USA, 1000-fold diluted), or β-actin (A5441, Sigma–Aldrich, 10,000-fold diluted). The secondary horseradish peroxidase-coupled goat anti-mouse-IgG (AP124P, Merck Millipore, Darmstadt, Germany) for β-actin antibody was 10,000-fold diluted and goat anti-rabbit-IgG (81-6120, Invitrogen Corporation, Camarillo, CA, USA) 2000-fold diluted for LC3 antibody. Signals of LC3 and β-actin proteins were developed with Western Lightning Chemiluminescence Reagent (Perkin–Elmer Life Sciences, Inc., Boston, MA, USA) and then digitalized with UnSCAN-IT software Automated Digitizing System, version 5.1.
β actin
Manufactured by PerkinElmer
Sourced in United States
β-actin is a highly conserved cytoskeletal protein that plays a crucial role in various cellular processes. It is commonly used as a reference gene or control for gene expression studies.
Automatically generated - may contain errors
Lab products found in correlation
Western lightning chemiluminescence reagent, by PerkinElmer (1 mentions)
A5441, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
L7543, by Merck Group (1 mentions)
Goat anti mouse igg ap124p, by Merck Group (1 mentions)
Goat anti rabbit igg 81 6120, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Enhanced chemiluminescence kit, by PerkinElmer (1 mentions)
Horseradish peroxidase labeled secondary antibody, by Abcam (1 mentions)
Cell lysis solution, by Merck Group (1 mentions)
β actin, by Proteintech (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
4 protocols using β actin
1
Western Blot Analysis of Autophagy Markers
2
Western Blot Analysis of Rab3D Protein
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Cells in the logarithmic growth period were lysed with cell lysis solution (Sigma-Aldrich, St. Louis, MO, USA). The supernatant was collected after they were centrifuged at 4°C (1000 rpm) for 5 mins. Total proteins were extracted and protein concentration was determined using BCA. Proteins (50 μg per lane) were separated using 12% SDS-PAGE. Proteins were then electrotransferred to a PVDF membrane (Amersham Biosciences, Piscataway, NJ, USA). The PVDF membrane was rinsed with TBS for 10–15 mins, placed in TBS/T blocking buffer containing 5% (w/v) skimmed milk powder. It was incubated at room temperature for 2 hrs following the addition of an appropriate dilution of primary antibodies (1:1000 Rab3D, Proteintech, Rosemont, IL, USA; 1:2000 β-actin, Proteintech, Rosemont, IL, USA). The membrane was then rinsed with TBST three times (5–10 mins/wash) and then incubated at room temperature for 1 hr with horseradish peroxidase-labeled secondary antibody (1:50,000; Abcam, Cambridge, UK; diluted with TBST containing 0.05% (w/v) skimmed milk powder). The membrane was then rinsed three times with TBST (5–10 mins/wash). Protein bands were detected using an enhanced chemiluminescence kit (Perkin-Elmer Inc.) and quantified as the ratio to β-actin. Quantification was performed using Imagequant LAS4000 (GE Healthcare, Japan).
3
KIF18A Protein Expression in NSCLC
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The expression of the Kif18A protein was detected by western blot. The specimens were homogenized on ice using a Potter Elvehjem glass homogenizers. The mixtures were mixed with RIPA lysis buffer and incubated on ice for 30 minutes. The lysates were collected, centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatants were used for the experiments. The protein concentrations were determined using the BCA assay. Total proteins (50 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred on nitrocellulose membranes. The membranes were blotted with antibodies against Kif18A (dilution: 1:500; Bioss Antibodies, Inc, Woburn, MA) and β-actin (dilution: 1:1000; Sigma US). The X-ray films were photographed using a gel imaging analysis system (LY-SUPER HP CCD camera system; Chengdu Liyang Precision Machinery Co, Ltd, Chengdu, China). The gray-scale value of each band was analyzed using the Lab Works 2.0 software (Perkin-Elmer Life Sciences, Waltham, MA), with β-actin as the internal reference. The relative expression of the Kif18A protein in each group was calculated according to the gray-scale value of the Kif18A band gray-scale value to that of β-actin. Each sample of NSCLC was tested for KIF18A along with its corresponding paracancerous normal tissue sample. The expression of KIF18A in NSCLS was compared with that of the normal tissue.
4
Quantifying Cellular mRNA Levels by smFISH
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Cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 70% ethanol for at least 2 hr at 4°C. After washing with wash buffer (2xSSC, 10% formamide), β-actin, MHCα or MLC2a MAGIC Probes were added at 37 nM and Stellaris β-actin, MHCα or MLC2a single-molecule FISH (smFISH) probes (Biosearch Technologies) were added at 125 nM to cells in hybridization buffer (2xSSC, 10% dextran sulfate, 1 μg/μl t-RNA, 0.02% BSA, 10% formamide, 40U Superase In RNase inhibitor) for 4 hr at 37°C. Cells were then washed with wash buffer and equilibrated with EMSA buffer. 50 ng purified MAGIC Factor was added to cells in EMSA buffer and reacted for 1 hr at 4°C. Cells were then washed with EMSA buffer and imaged. In RNA-FISH experiments, MAGIC Factor was labeled with Alexa Fluor 488, MAGIC Probe with Alexa Fluor 546 and smFISH probe with Quasar 670 dye.
For quantification of transcript levels, 3D images of cells stained with the β-actin, MHCα or MLC2a smFISH probes were acquired and transcripts levels quantified using Volocity software (Perkin Elmer) as previously described (Lifland et al., 2011 (link); Santangelo et al., 2009 (link)).
For quantification of transcript levels, 3D images of cells stained with the β-actin, MHCα or MLC2a smFISH probes were acquired and transcripts levels quantified using Volocity software (Perkin Elmer) as previously described (Lifland et al., 2011 (link); Santangelo et al., 2009 (link)).
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