After the behavioral assessment, the contusion cortex proteins around the injury edge were extracted (3 × 3 × 5 mm (width × deep × length)) and protein concentration was confirmed via Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein levels of ferroptosis-associated proteins GPX4, PTGS2, SLC7A11, and pathway-related proteins HO-1, NQO1, and Nrf2 were determined using western blotting. The primary antibodies for western blotting included anti-GPX4 (1:1000, Abcam, ab32503), anti-PTGS2 (1:1000, Abcam, ab59348), anti-SLC7A11 (1:1000, Cell Signaling Technology, USA, 8242), anti-NRF2 (1:500, Abcam, ab214185), anti-HO-1 (1:500, Abcam, ab175449), anti-NQO1 (1:1000, Novusbio, USA, NB10056565), as well as anti-GAPDH (1:2000, Proteintech, 60004-1-lg). After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proteins of all samples were transferred to nitrocellulose filter membrane. For binding primary antibody to secondary antibody, membrane experienced incubation by secondary antibody horseradish peroxidase-labeled goat anti-rabbit or goat anti-mouse for 60 min under ordinary temperature. Western blot results were analyzed using enhanced chemiluminescence.
Ab59348
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom
Ab59348 is a primary antibody produced by Cell Signaling Technology. It is designed for use in various research applications, such as Western blotting, immunohistochemistry, and immunofluorescence. The antibody specifically recognizes its target protein and can be used to detect and study its expression and localization in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab32503, by Abcam (2 mentions)
Ab214185, by Abcam (2 mentions)
Ab175449, by Novus Biologicals (2 mentions)
Nb10056565, by Proteintech (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bcl 2, by Abcam (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Histone 3, by Abcam (1 mentions)
Ai600 imaging system, by GE Healthcare (1 mentions)
Ace h3, by Merck Group (1 mentions)
H3k9ace, by Cell Signaling Technology (1 mentions)
Ab52894, by Cell Signaling Technology (1 mentions)
Hvlp04700, by Merck Group (1 mentions)
Imagej, by Bio-Rad (1 mentions)
A53227, by Thermo Fisher Scientific (1 mentions)
Ripa lysate, by Thermo Fisher Scientific (1 mentions)
Ab38449, by Abcam (1 mentions)
4 protocols using ab59348
1
Contusion Cortex Protein Profiling
2
Quantifying Ischemic Protein Changes
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Western blotting was used to determine the quantity of target proteins being made in the ischemic hemisphere. Briefly, brain tissues were milled in liquid nitrogen, and proteins were extracted using RIPA cleavage buffer. Protein intensity, with BSA as a standard, was measured according to the Bradford method. Each sample was separated using 12% SDS-PAGE and transferred onto a methanol-activated PVDF membrane (Millipore, Burlington, MA, USA) by wet electrotransfer. The membranes were blocked for 1.5 h at room temperature with 5% BSA. The membranes were then incubated with primary antibodies against Ace-H3 (Millipore, cat #9717, 1:1000), H3K9ace (Cell Signaling Technology, cat #6949, 1:1000), H3K27ace (Cell Signaling Technology, cat #4353, 1:1000), Histone3 (Abcam, ab1791, 1:1000), Bcl-2 (Abcam, ab59348, 1:1000), Bax (Cell Signaling Technology, cat #2772, 1:1000), caspase-3 (Cell Signaling, cat #9662, 1:1000), cleaved caspase-3 (Cell Signaling, cat #9664S, 1:1000) and GAPDH (SAB, cat #21612S; 1:1000). The total amount of ECL liquid was absorbed in a 1:1 ratio (solution A: B) to uniformly cover the entire film and was observed using an AI600 imaging system (GE Healthcare, USA). GAPDH was used as an internal reference for comparison of grayscale values.
3
Quantitative Protein Analysis in HeLa Cells
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Total protein concentration was extracted from HeLa cells using a RIPA lysate (89901, Thermo Fisher Scientific, Waltham, MA, USA). Protein concentration was measured using a protein detection kit (A53227, Thermo Fisher Scientific, Waltham, MA, USA). A volume of 30 μg protein for each cell was transferred to a PVDF membrane (HVLP04700, Millipore, Billerica, MA, USA) using the SDS-PAGE method. After the transfer was completed, the membrane was soaked from bottom to top with TBS, then placed in an incubation box containing a 5% skimmed milk powder solution (37°C, 1 h), incubated with shaking at room temperature on a decolorization shake flask for 2 hours, and then incubated with phosphorylated (p)-Akt (1: 1000; 56 KD; ab38449; Abcam, Cambridge, UK), Akt (1: 500; 55 KD; ab8805), Bax (1: 1000; 21 KD; ab32503), Bcl-2 (1: 1000; 26 KD; ab59348), Wnt1 (1 μg/mL; 41 KD; ab85060), MMP-9 (1 μg/mL; 95 KD; ab73734), EGFR (1: 1000; 134 KD; ab52894), β-catenin (1: 1000; 92 KD; #9562; Cell signaling technology, Danvers, MA, USA), or β-actin (1: 1000; 45 KD; #4970) overnight at 4°C. The target band was incubated with a goat anti-rabbit IgG H&L (HRP) (1: 5000; ab205718; Abcam, Cambridge, UK) for 2 h. Finally, the signals were detected using SignalFir ECL reagent (#6883) and the gray value of the band was analyzed and calculated using ImageJ (version 5.0, Bio-Rad, Hercules, CA, USA) [17 (link)].
4
Pericontusive Protein Expression Analysis
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Pericontusive cortex (3 × 3 × 5 mm (width × depth × length) surrounding the edge of the lesion) proteins were extracted (n = 6), and protein levels of Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), NF-κB, NLRP3, apoptosis-associated speck-like protein (ASC), and caspase-1 were measured by western blotting. Protein concentrations from tissue lysates were measured using the bicinchoninic acid method (cat. no. PA115; Tiangen Biotech). Primary antibodies for western blotting were anti-Bax (1:1000, Abcam, ab32503), anti-Bcl-2 (1:1000, Abcam, ab59348), anti-NF-κB (1:1000, Cell Signaling Technology, 8242), anti-NLRP3 (1;500, Abcam, ab214185), anti-ASC (1:500, Abcam, ab175449), anti-caspase-1 (1:1000, Novusbio, NB100-56565), and anti-GAPDH (1:2000, Proteintech, 60004-1-lg). Following sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the protein from each sample was transferred to the polyvinylidene fluoride (PVDF) membrane. To make the primary antibodies bind to the secondary antibodies, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000, Zsgb-Bio) for 60 minutes at room temperature. Western blot results were analyzed using Image Lab Software (version 3.0).
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