Protein samples were harvested from the brainstem of the alligator, chicken, and gerbil, as well as from the olfactory bulb of the mouse and the cortex of the rat. All samples were sonicated in Tris buffer (20 mM Tris pH 7.5, 150 mM NaCl, and 2 mM EDTA) with protease inhibitor (Sigma, St. Louis, MO) and centrifuged at 2300 xg for 10 min. The supernatant was collected and sampled for Bradford protein assay (Bio-Rad, Hercules, CA). Each sample (10 μg or 30 μg protein) was diluted in 6× Laemli buffer (300 mM Tris pH 6.8, 600 mM DTT, 12% SDS, 0.6% bromophenol blue, and 60% glycerol), incubated at 65°C for one hour, subjected to SDS-PAGE on 10% gel, and transferred to PVDF membrane. Membranes were incubated in 5% milk in phosphate buffered saline (PBS) with 0.1% Tween for one hour, primary antibody solution (anti-FMRP, 1:2000; anti-β-actin, 1:1000) for 2 hours, and secondary antibody solution (HRP-conjugated donkey-anti-rabbit, 1:200,000; Jackson ImmunoResearch Laboratories, West Grove, PA) with 2% milk for one hour. The blots were then developed with Immobilon chemiluminescent HRP substrate (Millipore, Billerica, MA) and X-ray film. Blots were stripped with Restore buffer (Bio-Rad) between different primary antibody stains.
Anti β actin
Manufactured by Jackson ImmunoResearch
Sourced in United States, United Kingdom
Anti-β-actin is a primary antibody that specifically binds to the β-actin protein, a ubiquitous cytoskeletal protein found in eukaryotic cells. It can be used for the detection and quantification of β-actin in various sample types using techniques such as Western blotting, immunocytochemistry, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon chemiluminescent hrp substrate, by Merck Group (1 mentions)
Hrp conjugated donkey anti rabbit, by Jackson ImmunoResearch (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Sds page, by Solarbio (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Sd 001, by Invent Biotechnologies (1 mentions)
Anti hif 1α, by Proteintech (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Anti pkr, by Abcam (1 mentions)
Anti ns1, by GeneTex (1 mentions)
Imagequant las 4000, by GE Healthcare (1 mentions)
Anti np, by GeneTex (1 mentions)
Anti flag, by Signalway Antibody (1 mentions)
Anti β actin, by Signalway Antibody (1 mentions)
Ecl detection reagent, by Cytiva (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Anti phospho enos ser1177, by Cell Signaling Technology (1 mentions)
6 protocols using anti β actin
1
Protein Expression Across Species
2
Quantitative Protein Analysis of Glioma Cells
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Total protein was extracted from glioma cells with lysis buffer (SD-001, Invent), and quantified with a BCA protein assay kit (Pierce). To perform Western blot analysis, equal amount of protein (20-40 μg) of each sample was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE, Solarbio) and transferred onto polyvinylidene difluoride membranes (PVDF, Millipore). PVDF was blocked with TBST containing 5% non-fat milk at room temperature for 1h, and then incubated with diluted primary antibodies at 4°C overnight, including anti-HIF1α (1:1000, Proteintech), anti-NCAN (1:1000, Abcam), anti-RhoA and anti-Rho-associated protein kinase (ROCK) (1:1000, Bioss), anti-β-actin(1:5000, Jackson), and anti-GAPDH (1:5000, Proteintech). The next day, the membranes were incubated with horseradish peroxidase conjugated goat anti rabbit or rabbit anti-goat secondary antibody (1:20000, Jackson) at room temperature for 1h, and then reacted with an enhanced ECL substrate (Pierce). Beta-actin was used as a control according to the protocol.
3
Western Blot Analysis of Viral Proteins
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Whole cell lysates were prepared and separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) followed by electro-transfer onto nitrocellulose (NC) paper (Bio-Rad). The filter was blocked in 5% skim milk for one hr, and then incubated with the primary antibodies at 4°C for overnight. Subsequently, the filter was rinsed with PBS containing 0.05% Tween 20 (PBS-T) for five times, followed by incubation with the corresponding secondary antibody conjugated with horseradish peroxidase (HRP) for 1 hr. After washing with PBS-T, protein signals were detected by enhanced chemiluminescence (ECL) and acquired by ImageQuant LAS 4000 (GE Healthcare, Uppsala, Sweden). The dilutions of each antibody were as follows: anti-β-actin (1:1,000; Signalway Antibody), anti-FLAG (1:2,500; Signalway Antibody), anti-β-actin (1:5,000; Jackson), anti-NS1 (1:2,000, GeneTex), anti-NP (1:1,000; GeneTex), anti-PKR (1:2,000, Abcam), anti-PKR-p (T446; 1:2,000; Abcam), anti-HA (1:2,500; Yao-Hong Biotechnology, Taiwan).
4
Protein Expression Analysis of Skin Samples
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Total protein lysates of skin samples were prepared with T-PER tissue protein extraction reagent (Pierce, USA). The prepared protein was subjected to SDS-polyacrylamide gel electrophoresis and subsequently transferred onto PVDF membranes (Bio-Rad, USA). The membranes were incubated with primary antibodies including anti-Bcl-2 (1 : 500), anti-Bax (1 : 500), anti-β-actin (1 : 1000), anti-p53 (1 : 500), anti-Caspase-3 (1 : 500), anti-Active Caspase-3 (1 : 500), anti-Caspase-9 (1 : 500), anti-Procaspase-9 (1 : 500), followed by incubation with appropriate HRP-conjugated secondary antibodies (Jackson ImmunoResearch, USA). All primary antibodies were purchased from Cell Signaling Technology. The protein bands were eventually visualized using an enhanced chemiluminescence (ECL) detection reagent (Amersham Pharmacia, USA).
5
Western Blot Analysis of Protein Expressions
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Protein lysates were prepared in RIPA lysis buffer with protease inhibitor cocktail (Roche), lysates were separated by SDS-PAGE, and the proteins were transferred to a PVDF membrane (Millipore). The membrane was reacted with the following primary antibodies at 1:1000: anti-p53 antibody (DO1) (Santa Cruz, sc-126), anti-eNOS (BD Transduction, 610296), anti-phospho-eNOS (ser1177) (Cell Signaling Technology, CST9571), anti-β-actin (Cell Signaling Technology, 4970), and anti-α-tubulin (Cell Signaling Technology, 2125). Blocking was done with 5% skim milk. The secondary antibodies were horseradish peroxidase-conjugated anti-mouse immunoglobulin-G (Jackson, 115-035-003) for anti-p53 and anti-eNOS, as well as horseradish peroxidase-conjugated anti-rabbit immunoglobulin-G (Jackson, 115-035-144) for anti-phospho-eNOS, anti-β-actin and anti-α-tubulin. Secondary antibodies were added at a concentration of 1:5000. Proteins were detected by chemiluminescence using ECL western blotting detection reagents (GE Healthcare, UK). Relative protein levels were quantified with Image J software and were normalized by using β-actin or α-tubulin as loading controls.
6
Modulation of Stress Signaling Pathways
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B16-F10 cells and HaCaT cells were plated in 24-well plates and grown for 24 h. Then both types of cells were incubated with various concentration of DHG for 1 h at 37°C, but only B16-F10 cells were under UV (100 mJ) stress. Extracted protein samples from each treatment group (containing 50 μg of protein) were separated on 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to immobile polyvinylidene difluoride membranes (Millipore, Billerica, MA). The membranes were incubated for 1 h with 5% dry skim milk in TBST buffer (0.1M Tris-HCl, pH 7.4, 0.9% NaCl, 0.1% Tween-20) to block non-specific binding. Then, they were incubated with rabbit antibodies against AKT, p-JNK, p-ERK, p-p38 (Abcam, Cambridge, UK), and anti-β-actin (Jackson, West Grove, PA, USA). Subsequently, the membranes were incubated with the conjugated affinity goat antirabbit IgG (Jackson). Expression of these proteins was detected by a chemiluminescence detection system according to the manufacturer's instructions (ECL, Amersham, Berkshire, UK) [10] .
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