Nuclear extract proteins were dissolved in Laemmli sample buffer (62.5 mmol/L Tris-HCl, pH 6.8, 50 mmol/L DTT, 2% w/v SDS, 20% w/v glycerol, 0.2% w/v bromophenolblue) and separated by SDS-PAGE (10 or 18% T; BioRad mini protean III cell; BioRad Laboratories GmbH, Munich, Germany). Based on label free quantification [29 (link)–31 (link)], the protein amounts were adjusted among the samples, separated by SDS-PAGE, and blotted onto low fluorescent polyvinylidenedifluoride (PVDF) membranes (Mini Trans-Blot® Cell, BioRad Laboratories). Membranes were blocked overnight (4°C, Immunoblot Blocking solution, AdvanBlock, Advansta), incubated with primary antibodies (1:10,000; in blocking buffer, 1 h, RT; rabbit polyclonal anti-Nrf2, rabbit polyclonal anti-NFκB p65 (pSer 536) [Santa Cruz Biotechnology, Heidelberg, Germany] or mouse polyclonal anti-H2AX (pSer 139) [Thermo Fisher Scientific, München, Germany]) and washed (Immunoblot Washing solution, AdvanWash, Advansta). Peroxidase-conjugated donkey anti-rabbit (1:2,500; in blocking buffer) or anti-mouse Ab (1:10,000; in blocking buffer) was added (1H, RT). Membranes were washed (Immunoblot Washing solution, AdvanWash, Advansta), WesternBright Sirius HRP substrate (Advansta) added, and the blot imaged on a Fusion FX7 Imaging system (PeqlabBiotechnologie GmbH, Erlangen, Germany).
Biorad mini protean 3 cell
Manufactured by Bio-Rad
Sourced in Germany
The Bio-Rad Mini-PROTEAN III Cell is a vertical electrophoresis system used for the separation of proteins based on their molecular weight. It accommodates 1 or 2 mini-gel cassettes and is designed for efficient and consistent protein separation.
Automatically generated - may contain errors
Lab products found in correlation
Westernbright sirius hrp substrate, by Advansta (2 mentions)
Advanwash, by Advansta (2 mentions)
Immunoblot washing solution, by Advansta (2 mentions)
Mini trans blot cell, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
β actin, by BD (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Polyvinylidene fluoride pvdf membrane, by GE Healthcare (1 mentions)
Ripa lysis buffer, by Santa Cruz Biotechnology (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using biorad mini protean 3 cell
1
Western Blot Analysis of Nuclear Proteins
2
SDS-PAGE Analysis of Protein Molecular Weights
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Molecular weight distribution was determined by SDS-PAGE. All of the samples were dissolved in a Laemmli sample buffer containing 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 25% (v/v) glycerol, 0.01% bromophenol blue, and 5% β-mercaptoethanol, then heated in a 90–95 °C water bath for 5 min [31 (link)]. Samples were separated on a 4% stacking and 16.5% separating gel (Bio-Rad Laboratories, Hercules, CA) using Tris/Glycine/SDS running buffer (25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS) in a Bio-Rad Mini-PROTEAN III Cell (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The amount that was loaded to the wells was adjusted to 1 mg for the hydrolysates, 0.5 mg for the control, and 10 µL of a protein molecular weight standard (Precision Plus Protein™ Dual Xtra Prestained Protein Standards 2-250 kD, Bio-Rad Laboratories, Inc., Hercules, CA, USA). Separated proteins were fixed in a gel fixing solution containing 50% (v/v) ethanol and 10% (v/v) acetic acid then washed with a solution consisting of 50% (v/v) methanol and 10% (v/v) acetic acid. Following washing, gel staining was performed using Coomassie blue R-250 solution at room temperature for 3 h with gentle agitation. (Bio-Rad Laboratories, Inc., Hertfordshire, UK), then the gel was destained with washing solution until the background color was removed.
3
Oxidized Protein Detection by DNPH Derivatization
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Proteins were dissolved in sample buffer (62.5 mmol/L TrisHCl, pH 6.8, 50 mmol/L DTT, 2% w/v SDS, 20% w/v glycerol, 0.2% w/v bromophenol blue) and separated by SDS-PAGE (10% T; BioRad mini protean III cell; BioRad Laboratories GmbH, München, Germany). Proteins were semidry blotted onto a low fluorescent polyvinylidene difluoride (PVDF) membrane (Trans-Blot Turbo Transfer System, BioRad Laboratories GmbH, München, Germany). Membranes were equilibrated (2 M HCl), derivatized with DNPH (1 g/L in 2 M HCl, 30 min, RT), washed with 2 M HCl (5 min) and methanol (5 min, 5 times). Membranes were blocked overnight (4 °C, Immunoblot Blocking solution; AdvanBlock, Advansta), incubated with goat anti-DNP Ab (1:10,000; in blocking buffer, 1 h, RT), and washed (Immunoblot Washing solution, AdvanWash, Advansta) before peroxidase-conjugated donkey anti-goat Ab (1:10,000, in blocking buffer) were added (1 h, RT). Membranes were visualized using WesternBright Sirius HRP substrate (Advansta) and imaged on a Fusion FX7 Imaging system (Peqlab Biotechnologie GmbH, Erlangen, Germany).
4
Extraction and Analysis of Nuclear Proteins
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Whole-cell lysates were prepared in RIPA buffer and nuclear extracts were prepared as previously described [15] (link) with slight modifications [16] (link). In brief, nuclear pellets were resuspended in 3 mL of 0.25 M sucrose containing 10 mM MgCl2, layered over with 3 mL cushion of 0.35 M sucrose containing 0.5 mM MgCl2 and centrifuged at 1400g for 5 min at 4 °C. The resulting nuclear pellets were resuspended in ice-cold buffer containing 20 mM HEPES pH 7.9, 400 mM NaCl, 1 mM each of DTT, EDTA, EGTA and PMSF, placed on a rotatory shaker for 15 min followed by centrifugation at 14,000 rpm for 10 min.
Aliquots of the protein extracts were subjected to the western blot analysis (whole cell extracts in RIPA buffer) or separated by SDS-PAGE (10% T; BioRad mini protean III cell; BioRad Laboratories GmbH, München, Germany) for further in-gel digestion (nuclear-enriched extracts). Western blot analysis was performed using following primary antibodies: phospho-specific (Ser139) and total histone H2AX (Cell signaling Technology) and β-actin (BD Biosciences). Secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA). Total and phosphorylated histone H2AX levels were normalized to β-actin. Western blots were developed using chemiluminescence detection and analyzed by densitometry.
Aliquots of the protein extracts were subjected to the western blot analysis (whole cell extracts in RIPA buffer) or separated by SDS-PAGE (10% T; BioRad mini protean III cell; BioRad Laboratories GmbH, München, Germany) for further in-gel digestion (nuclear-enriched extracts). Western blot analysis was performed using following primary antibodies: phospho-specific (Ser139) and total histone H2AX (Cell signaling Technology) and β-actin (BD Biosciences). Secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA). Total and phosphorylated histone H2AX levels were normalized to β-actin. Western blots were developed using chemiluminescence detection and analyzed by densitometry.
5
Protein Quantification and Western Blot
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Cells were scraped and lysed in RIPA lysis buffer (sc24948, Santa Cruz Biotechnology, Dallas, TX). Protein concentration of the lysed samples were measured by the Pierce® BCA Protein Assay Kit (Thermo Fisher Scientific). Electrophoresis was done using premade Biorad stain-free gels (Biorad Mini-PROTEAN 3 Cell), and the protein was transferred to polyvinylidene fluoride PVDF membranes (GE Healthcare, Little Chalfont, U.K) by BioradTurbo Transfer System. Before stained with respective antibodies (Additional file 2 : Table S3), total protein was assessed by imaging on ChemiDocTM XRS+ with Image Lab Software (Biorad).
6
Structural Integrity of Encapsulated pIL-1β
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The primary structural integrity of released pIL-1β from ALG and ALG/CHI MPs after 1 day and 6 days in PBS (pH 7.4) was determined by SDS–PAGE and compared with pure pIL-1β and molecular weight markers. pIL-1β was present at 5 ug in both unencapsulated (pure) and encapsulated ALG and ALG/CHI MP samples. Aliquots (15 µL) of these dispersions were mixed with 4× sample buffer (0.25 mol/L Tris-HCl, pH 6.8, 80 g/L SDS, 200 mL/L glycerol, 100 g/L β-mercaptoethanol, and 1 g bromophenol blue) [82 (link)] denatured at 100 °C for 3 min. The gel (12% (w/v) acrylamide) was run under reducing conditions using Bio-Rad Mini-PROTEAN 3 Cell (Bio-Rad Laboratories) at a constant voltage mode of 120 V in a Tris/glycine/SDS buffer. The gel was stained with Coomassie blue staining solution and destained with 5% (v/v) acetic acid solution overnight.
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