The antiserum directed towards Sepp (1:400) was generated in rabbits by immunization with a synthetic C-terminal peptide (ImmunoGlobe, Himmelstadt, Germany) and its specificity verified using wild-type and Sepp-knockout mice [22 (link), 25 (link)]. For serum Sepp quantification, 0.2 μl serum was applied per lane. 25–100 μg protein from the cytosolic fraction were separated on SDS/12% polyacrylamide gels. Antibodies against Sepk and Seps were from Sigma (ATLAS Prestige antibodies, rabbit polyclonal) and used at 1:500, 1:1000 dilution, respectively. Rabbit polyclonal antibodies against Gpx1 and Gpx4 were both from Abcam (Cambridge, UK) and were used at 1:2000 and 1:1000, respectively. The polyclonal rabbit Sepsecs antiserum (Sigma, München, Germany) was used at 1:1000 dilution. Chicken polyclonal antibodies directed towards SECp43 were kindly provided by Dr. Paula Grabowski (University of Pittsburgh) and used at a 1:1000 dilution. After electrotransfer, nitrocellulose membranes were stained with Ponceau Red, photographed and blocked with 5% BSA for 1 hour at 25°C. Rabbit polyclonal β-actin antiserum (Sigma, Munich, Germany) was used as a loading control at 1:2000 dilution.
β actin antiserum
Manufactured by Merck Group
Sourced in Germany, United Kingdom, United States
β-actin antiserum is a laboratory reagent used to detect and measure the presence of the β-actin protein in biological samples. It is a polyclonal antibody that specifically binds to the β-actin protein, allowing for its identification and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Bm chemiluminescence western blot kit, by Roche (1 mentions)
Molecular imaging software, by Kodak (1 mentions)
Ndrg1, by Thermo Fisher Scientific (1 mentions)
Genetools of chemigenius, by Syngene (1 mentions)
Ne pertm nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions)
Ndrg2, by Abcam (1 mentions)
Ndrg3, by Abcam (1 mentions)
Maspin, by BD (1 mentions)
Hif 2α, by Novus Biologicals (1 mentions)
Hif 1α, by BD (1 mentions)
4 protocols using β actin antiserum
1
Quantification of Selenoproteins by Western Blot
2
Western Blot for NHE8 and β-catenin
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Total protein was prepared in a RIPA buffer. For Western blot detection, NHE8 antibody was used in a 1:2000 dilution to detect NHE8 protein,5 (link) β-catenin antiserum (catalog no. ab32572; Abcam, Cambridge, MA) was used in a 1:4000 dilution to detect β-catenin protein, and β-actin antiserum (A5316; Sigma) was used in a 1:5000 dilution to detect β-actin protein. Western detection was performed using the BM Chemiluminescence Western Blot Kit (catalog no. 11 520 709 001; Roche Diagnostics, Indianapolis, IN).
3
Western Blot Analysis of Liver Tissue
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Liver tissues were homogenized in lysis buffer (0.6% NP-40, 150 mM NaCl, 10 mM HEPES (pH 7.9), 1 mM EDTA, and 0.5 mM PMSF) at 4°C. Fifty microgrammes of protein subjected to 10 % SDS-PAGE was transferred to polyvinylidene fluoride (PVDF) membrane (NEN Life Science, Boston, MA), blot immunodetected with enhanced chemiluminescence (ECL) Western blot kit (Amersham International, Amersham, UK) in which goat anti-mouse COX-2, nuclear factor-kappaB (NF-κB), inducible nitric oxide synthase (iNOS) and β-actin antiserum served as primary antibody (Millipore, MA) and goat anti-rabbit IgG-biotinylated species-specific whole antibody (Calbiochem) was used as secondary antibody. Blots were quantified by relative intensity compared to control with Kodak Molecular Imaging Software (Version 4.0.5, Eastman Kodak Company, Rochester, NY), represented in relative intensities.
4
Nuclear and Cytoplasmic Protein Extraction
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For nuclear and cytoplasmic extraction, cells were cultured in an RPMI-1640 medium with 10% FCS for 48 h, and then harvested with trypsin, and washed twice with PBS. Nuclear and cytoplasmic fractions were separated using the NE-PERTM Nuclear and cytoplasmic extraction kit (Thermo, Rockford, NJ, USA) as described by the manufacturer. Equal amounts of whole cell, nuclear, or cytoplasmic lysis were loaded onto a 10% SDS-polyacrylamide gel and assayed by enhanced chemiluminescence as described by the manufacturer (PerkinElmer Inc, Waltham, MA, USA). Blotting membranes were probed with antiserum of MT3 (Sigma-Aldrich Co.), heme oxygenase-1 (HO-1; Stressgen, Victoria, BC, Canada), NDRG1 (Invitrogen), NDRG2, NDRG3 (Abcam, Cambridge, UK), HIF-1α, MASPIN (BD Biosciences, San Jose, CA, USA), HIF-2α (Novus, Littleton, CO, USA), or β-actin antiserum (Millipore, Billerica, MA, USA). The intensities of different bands were analyzed using the GeneTools of ChemiGenius (Syngene, Cambridge, UK).
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