Plates of starved worms were chunked onto 15 cm plates and allowed to grow for 3 days, with additional food added at day 2 to prevent starvation. Worms were collected from NGM plates using 1X M9 buffer (0.04 M Na2HPO4, 0.02 M KH2PO4, 0.009 M NH4Cl, 0.02 M NaCl), washed with extract buffer (50 mM HEPES [pH 7.4]; 70 mM K-Acetate, 5 mM Mg-Acetate, 0.05% NP-40, and 10% glycerol) and frozen at —80°C. A cold motor and pestle were used to make worm lysates from the frozen pellets. The total protein concentration of the lysates was quantified using a Bradford assay (Sigma-Aldrich) and an equivalent amount of lysates from each strain were subjected to SDS-PAGE and immunoblotting with a custom ADR-2 antibody (described in Deffit et al., 2017 (link)) and an antibody to β-actin (Cell Signaling Technology).
Antibody to β actin
Manufactured by Cell Signaling Technology
Antibody to β-actin is a lab equipment product that can be used to detect and quantify the presence of the β-actin protein in biological samples. β-actin is a widely expressed cytoskeletal protein that plays a fundamental role in various cellular processes. This antibody provides a specific and reliable way to measure β-actin levels in research applications.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey imaging system, by LI COR (4 mentions)
Bradford assay, by Merck Group (2 mentions)
Criterion tgx precast gel, by Bio-Rad (2 mentions)
Fli 1 polyclonal antibody, by Cell Signaling Technology (2 mentions)
Iblot 2 transfer stack, by Thermo Fisher Scientific (2 mentions)
Criterion tgx stain free protein gel, by Bio-Rad (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Ab105472, by Abcam (1 mentions)
Amersham ecl prime, by GE Healthcare (1 mentions)
Ab93357, by Abcam (1 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Ab6789, by Abcam (1 mentions)
Protease and phosphatase inhibitor, by Roche (1 mentions)
8 protocols using antibody to β actin
1
Quantifying Worm Protein Levels
2
Western Blot Analysis of Fli-1 Protein
3
Quantifying Worm Protein Levels
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Plates of starved worms were chunked onto 15 cm plates and allowed to grow for 3 days, with additional food added at day 2 to prevent starvation. Worms were collected from NGM plates using 1X M9 buffer (0.04 M Na2HPO4, 0.02 M KH2PO4, 0.009 M NH4Cl, 0.02 M NaCl), washed with extract buffer (50 mM HEPES [pH 7.4]; 70 mM K-Acetate, 5 mM Mg-Acetate, 0.05% NP-40, and 10% glycerol) and frozen at —80°C. A cold motor and pestle were used to make worm lysates from the frozen pellets. The total protein concentration of the lysates was quantified using a Bradford assay (Sigma-Aldrich) and an equivalent amount of lysates from each strain were subjected to SDS-PAGE and immunoblotting with a custom ADR-2 antibody (described in Deffit et al., 2017 (link)) and an antibody to β-actin (Cell Signaling Technology).
4
Western Blot Analysis of Fli-1 and Actin
5
Protein Extraction and Western Blot Analysis
6
Protein Expression Analysis of Innate Immune Markers
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The joint and lung tissue lysates (20 μg per sample) were resolved on NuPage 4–12% Bis-Tris protein gels (Invitrogen) and transferred onto nitrocellulose membranes. The membranes were blocked overnight with 5% milk powder (w/v) and probed with antibodies for murine CRAMP (rabbit polyclonal, Abcam, USA, catalog number ab93357), S100A8 (rat monoclonal [clone ABM4A69], Abcam, catalog number ab220174), S100A9 (rat monoclonal [clone 2B10], Abcam, catalog number ab105472), α-defensin 1 (goat polyclonal, Abcam, catalog number ab122884), β-defenisn 2 (rabbit polyclonal, MyBioSource, USA, catalog number MBS2005685) and β-defensin 14 (rabbit polyclonal, MyBioSource, catalog number MBS1490249). Antibody to β-actin (Cell Signaling Technologies) was used to normalize for protein loading. Affinity-purified horseradish peroxidase (HRP)-linked secondary antibodies (Cell Signaling, USA) along with Amersham ECL Prime (GE Healthcare) was used for detection. The blots were imaged using AmershamTM Imager 680 blot and gel imager. Densitometry assessment of band intensity was determined using AmershamTM Imager 680 analysis software version 2.0. The relative band intensity was assessed after normalization with the band intensity for β-actin.
7
Western Blot Analysis of Fli-1 Expression
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Cells were lysed in Radioimmunoprecipitation assay (RIPA) lysis buffer. After lysis, the supernatant was collected. Equal amounts of protein (20 µg-40 µg) were run for 1.5 hours at 130V on a 4–20% Criterion TGX Stain-Free Protein Gel (Bio-Rad, Hercules, CA) and electrotransferred to a PVDF membrane by Iblot2 transfer stacks (Invitrogen, Waltham, MA). Transferred proteins were probed with both a Fli-1 polyclonal antibody described previously (20 (link)) and an antibody to β-actin (Cell Signaling, Beverly, MA). The results were visualized using the Odyssey Imaging System (LI-COR, Lincoln, NE).
8
Western Blot Protocol for HIF-1 and HO-1
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Cells were lysed in radioimmunoprecipitation assay lysis buffer (Sigma) supplemented with protease and phosphatase inhibitors (Roche), cleared by centrifugation at 13 000 rpm for 10 min, and analyzed using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific, no. 23225). Equal protein amounts were loaded, separated by sodium dodecyl-sulfate polyacrylamide gel electrophoresis, electrotransferred to nitrocellulose membranes, and blocked in 5% nonfat milk solution for 2 h. Membranes were probed with primary antibody, and signal was detected using appropriate horseradish peroxidase-conjugated secondary antibodies. All primary antibodies were used at 1:000 dilution and overnight incubation at 4 °C, followed by a 1-h incubation with horseradish peroxidase-conjugated secondary antibodies at 1:10 000 dilution at room temperature. Antibodies to HIF-1(no. ab179483) and HO-1 (no. ab13243) were from Abcam, and the antibody to β-actin was from Cell Signaling (no. 3700S). Secondary antibodies (no. ab97051 and ab6789) were from Abcam. Semiquantitative densitometry was carried out using Image J.
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