The protein content of Sirtuin3, a sensor of mitochondrial and metabolic balance, was determined by Western blot. Forty µg of total protein placental homogenate were separated using 7/13% SDS‐PAGE and transferred into nitrocellulose membranes (iBlot Gel Transfer Stacks; Life Technologies, Waltham, MA, USA). The membranes were hybridised with anti‐Sirtuin3 (44 KD; 1:250; Merck Millipore, Burlington, MA, USA) overnight and at 4°C. Sirtuin3 protein expression was normalised by β‐actin protein (47 KD; 1:30 000; Sigma‐Aldrich, St. Louis, MO). The ImageQuantLD program was used to quantify chemiluminescence and the results were expressed as the Sirtuin3/β‐actin ratio.
β actin protein
Manufactured by Merck Group
Sourced in United States
β-actin protein is a highly conserved cytoskeletal protein that is involved in the maintenance of cell structure and motility. It is a component of the actin filaments that make up the cytoskeleton of eukaryotic cells. The β-actin protein plays a crucial role in various cellular processes, including cell division, intracellular transport, and signal transduction.
Automatically generated - may contain errors
Lab products found in correlation
Iblot gel transfer stack, by Thermo Fisher Scientific (2 mentions)
Lamin a c, by Merck Group (1 mentions)
Ecl detection system reagent, by Thermo Fisher Scientific (1 mentions)
P akt ser473, by Cell Signaling Technology (1 mentions)
P p70s6k thr389, by Cell Signaling Technology (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ab20654, by Abcam (1 mentions)
Ab109414, by Abcam (1 mentions)
P0260, by Agilent Technologies (1 mentions)
Ab2185, by Abcam (1 mentions)
Ab109268, by Abcam (1 mentions)
P rps6ser240 244, by Cell Signaling Technology (1 mentions)
Bnip3, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Lysis buffer, by Kurabo (1 mentions)
Amyloid precursor protein, by Abcam (1 mentions)
Cd206, by Abcam (1 mentions)
Cd163, by Abcam (1 mentions)
6 protocols using β actin protein
1
Quantifying Sirtuin3 Protein Expression
2
Western Blot Analysis of Transfected Cells
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Western blot analyses of the cells transfected with either of the vectors indicated above were performed essentially as described [34 (link)]. The protein samples were size fractionated using a gradient 12% SDS polyacrylamide gel, and a commercially available antibodies were used for the detection of the V5 peptide tag (Thermo Fisher Scientific) and β-actin protein (Sigma Chemical Co, St. Louis, MO).
3
Western Blot Analysis of TBI-Induced Signaling
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Western blot was performed on samples harvested 24 hours post‐TBI. Cytosolic and nuclear extracts were prepared as described previously.21 (link) The membranes were probed with specific Abs: anti‐NF‐κBp65 (1:500; sc:8008), anti‐iNOS (1:500; sc:8310), anti‐IκB‐α (1:500; sc:1643), anti‐COX2 (1:500; sc‐1746), anti‐IκB‐α (1:500; sc:1643), anti‐phospho‐mTOR (1:1000; #2971S), anti‐Bax (1:500 sc:7480) and anti‐Bcl‐2 (1:500 sc:7382), in 1 × PBS, 5% w/v non‐fat dried milk, 0.1% Tween‐20 at 4℃, overnight. β‐actin protein (cytosolic fraction 1:500; sc:8432) or lamin A/C (nuclear fraction 1:500 Sigma‐Aldrich Corp.) was used to samples normalization. Signals were detected with enhanced chemiluminescence (ECL) detection system reagent according to the manufacturer's instructions (Thermo). The relative expression of the protein bands was quantified using a standardization to β‐actin and lamin A/C levels. Images of blot signals (8 bit/600 dpi resolution) were imported to analysis software (Image Quant TL, v2003).
4
Protein Expression Analysis of Lysed Cells
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After treatments, cells were washed with ice-cold PBS and were lysed in a buffer containing 0.25 mM sucrose, 10 mM Tris and 1 mM EDTA with protease and phosphatase inhibitors (Roche Diagnostics, Basilea, Suiza) by sonication during 2 pulses of 20 s at 60% amplitude, and centrifuged for 10 min at 14,000 g. Equal amounts of protein were separated by SDS-PAGE, transferred to a PVDF membrane (Bio-Rad, Hercules, CA, USA) and were blotted with: HIF-1α (ab2185, Abcam, Cambridge, UK), HIF-2α (ab20654, Abcam), NIX (ab109414, Abcam), p-mTORSer2448 (ab109268, Abcam), BNIP3 (sc-56167, Santa Cruz Biotechnology), p-mTORSer2481 (sc-293132, Santa Cruz Biotechnology), LC3 (PM036, MBL International, Woburn, MA, USA), p62 (#5114, Cell Signaling, Beverly, MA, USA), p-RP-S6Ser240/244 (#5364, Cell Signaling), p-p70S6KThr389 (#9206, Cell Signaling) and p-AktSer473 (#4060, Cell Signaling) antibodies, using β-actin protein (A3854, Sigma) as loading control. After three PBS-T washes, membranes were incubated for 1 h at room temperature with the antirabbit (31460, Thermo Fisher Scientific) and antimouse (P0260, Dako, Glostrup, Denmark) secondary antibodies. Proteins were visualized using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific). Specific band density was measured with ImageJ software (National Institute of Mental Health, Bethesda, MD, USA).
5
Cerebrum Protein Analysis in Mice
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Cerebrum samples were collected from all mice 2 years after treatment and were homogenized in lysis buffer (Kurabo, Osaka, Japan) and centrifuged at 8000 × g for 10 min. Western blot analysis was performed using previously described methods.30 (link) The membranes were incubated at room temperature for 1 h with primary antibodies against amyloid precursor protein (1:1000; Abcam, Cambridge, MA, USA), amyloid-β (1:1000; Rockland Immunochemicals Inc., Gilbertsville, PA, USA), ionized calcium-binding adaptor molecule 1 (Iba1, marker of microglial and macrophage, 1:1000; Wako, Osaka, Japan), chemokine receptor 7 (CCR7, 1:1000; Abcam), CD80 (1:1000; Bioss Antibodies Inc., Woburn, MA, USA), CD163 (1:1000; Abcam), CD206 (1:1000; Abcam), plasmin (1:1000; Abgent, San Diego, CA, USA), urokinase-type plasminogen activator (uPA, 1:500; Abcam), and β-actin protein (1:5000; Sigma-Aldrich, St. Louis, MO, USA). The protein bands on the membranes were visualized following the incubation of membranes with horseradish peroxidase-conjugated secondary antibody (Novex, Frederick, MD, USA); band intensities were detected using the ImmunoStar Zeta regent (Wako, Osaka, Japan). The images of protein bands were acquired using the multi-grade software program (Fuji-film, Greenwood, SC, USA).
6
Protein Expression Profiling in Heart and Placenta
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Twenty to forty µg of total protein homogenate of heart and placenta from both cases and controls were separated using 7/13% SDS-PAGE and transferred to nitrocellulose membranes (iBlot Gel Transfer Stacks, Life Technologies, Waltham, MA, USA). The membranes were hybridized with specific antibodies overnight at 4ºC. The expression of all studied proteins was measured once and normalized to β-actin protein (47kDa; 1:30.000; Sigma-Aldrich, St.Louis, MO, USA) which was used as a loading control. Inter-blot control samples were used to evaluate membrane variability. The ImageQuantLD program was used to quantify chemiluminiscence.
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