Whole-cell lysates for western blotting were prepared as described [4 (link)], and protein lysates were resolved by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Invitrogen, Carlsbad, CA, http://www.invitrogen.com ). Membranes were blocked with 5% non-fat milk for 1 h at room temperature and subsequently incubated overnight at 4°C with primary antibodies at the following dilutions: JUN, p-JUN, FOS, p-FOS, JUNB 1:500 (60A8, D47G9, 9F6, D82C12, C37F9, respectively; all from Cell Signaling Technology, Danvers, MA, http://www.cellsignal.com ); Mn-SOD2 1:500 (06-984; Millipore, Billerica, MA, http://www.millipore.com ); p21 and p53 1:200 (sc-756, sc-99, respectively; Santa Cruz); β-actin 1:5000 (8226, Abcam, Cambridge, MA, http://www.abcam.com ); p16 1:500 (554079; BD Pharmingen, San Jose, CA, http://www.bdbiosciences.com ); PKD/PKCμ, Phospho-PKD/PKCμ (Ser916), Phospho-PKD/KCμ (Ser744/748) 1:1000 (2052, 2051, and 2054, respectively; Cell Signaling Technology), were used for SER 910 and 938/942 detection respectively; SRF 1:500 (NBP1-61263; Novus Biologicals, Littleton, CO, http://www.novusbio.com ). Signals were detected using the appropriate peroxidase-conjugated secondary antibody (Dako, Glostrup, Denmark, http://www.dako.com ). Western blots were scanned and quantified by densitometry using ImageJ software (NIH, Bethesda, MD, www.nih.gov ).
Mn sod2
Manufactured by Merck Group
Mn-SOD2 is a laboratory equipment product manufactured by the Merck Group. It is an enzyme that catalyzes the dismutation of superoxide radicals, converting them into oxygen and hydrogen peroxide. The core function of Mn-SOD2 is to provide an efficient means of managing oxidative stress in various research and experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
P fos, by Cell Signaling Technology (1 mentions)
Sc 756, by Santa Cruz Biotechnology (1 mentions)
β actin, by Abcam (1 mentions)
P jun, by Cell Signaling Technology (1 mentions)
Bcl xl, by Merck Group (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
γh2ax, by Merck Group (1 mentions)
Bcl 2, by Merck Group (1 mentions)
Phosphor akt ser473, by Cell Signaling Technology (1 mentions)
Collagen 1, by Santa Cruz Biotechnology (1 mentions)
Fibronectin, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Mcl 1, by Santa Cruz Biotechnology (1 mentions)
Gpx1 2, by Santa Cruz Biotechnology (1 mentions)
2 protocols using mn sod2
1
Western Blot Protein Detection Protocol
2
Protein Quantification and Western Blot Analysis of BMSC-ECM
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Cells were suspended in lysis buffer (20 mM Tris-HCl, pH 7.8, 50 mM NaCl, 5 mM EGTA, and 1% v/v Triton X-100) containing freshly added protease and phosphatase inhibitors (Roche). Lysates were clarified by centrifugation at 4°C, and protein concentration was determined by Bio-Rad protein assay.
To verify the protein contents of the BMSC-ECM after alkaline detergent extraction, BMSC-ECM were scratched from culture plate and dissolved in buffer containing 100 mM Tris-HCl (pH 6.8), 200 mM dithiothreitol and 4% SDS. Samples were homogenized by pass through 27 g needle for at least 7 times, incubated on ice for 60 minutes, cleared by centrifugation and subjected to protein concentration assessment. Then 0.2% glycerol and 0.2% bromophenol blue (final concentration) were added and samples were heated at 95°C for 5 minutes.
The following antibodies are used: phosphor-AKT Ser 473 and were from Cell Signalling. γ-H2AX, osteopontin, β-actin, MnSOD2, BCL-2, and BCL-xL were from Millipore. p27, p21, collagen 1, fibronectin, MCL-1, Gpx1/2, were from Santa Cruz Biotechnology.
To verify the protein contents of the BMSC-ECM after alkaline detergent extraction, BMSC-ECM were scratched from culture plate and dissolved in buffer containing 100 mM Tris-HCl (pH 6.8), 200 mM dithiothreitol and 4% SDS. Samples were homogenized by pass through 27 g needle for at least 7 times, incubated on ice for 60 minutes, cleared by centrifugation and subjected to protein concentration assessment. Then 0.2% glycerol and 0.2% bromophenol blue (final concentration) were added and samples were heated at 95°C for 5 minutes.
The following antibodies are used: phosphor-AKT Ser 473 and were from Cell Signalling. γ-H2AX, osteopontin, β-actin, MnSOD2, BCL-2, and BCL-xL were from Millipore. p27, p21, collagen 1, fibronectin, MCL-1, Gpx1/2, were from Santa Cruz Biotechnology.
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