Following completion of treatment, BEAS-2B cells were scraped, re-suspended in phosphate-buffered saline (PBS), and then lysed/homogenized to prepare for the assay. An equal amount of protein of BEAS-2B cell lysate was separated on 4–12% Bis-Tris gel, transferred to a PVDF membrane, and blocked with Starting Block (TBS) blocking buffer (ThermoFisher-Scientific, catalog number 37543). The membrane was incubated with mouse monoclonal anti-cathelicidin antibody (ThermoFisher-Scientific, MA5–18048, 1:1000) for 2 hours at room temperature. As an internal control, a mouse monoclonal anti-alpha-tubulin antibody was used (DSHB University of Iowa, 12G10, 1:3000). For imaging of cathelicidin (CAMP) and α-tubulin protein, the membrane was incubated with IRDye 800CW goat anti-mouse IgG (H+L) (Li-Cor Biosciences, P/N 926–32210, 1:15000) for 1 hour at room temperature. Afterward, the membrane was processed with the Odyssey CLx Imaging system (Li-Cor Biosciences). Densitometric analysis of bands was performed using Image Studio software (Li-Cor Biosciences).
Tbs blocking buffer
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, France
TBS) blocking buffer is a solution used in various laboratory techniques, such as Western blotting and ELISA, to prevent non-specific binding of antibodies or other proteins to the membrane or plate. It is a Tris-buffered saline solution that helps to minimize background signals and improve the specificity of the assay.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Irdye 800cw goat anti mouse igg h l, by LI COR (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Image studio software, by LI COR (1 mentions)
Completeultra mini tablets, by Roche (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti h3cit, by Abcam (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Iblot dry blotting system, by Thermo Fisher Scientific (1 mentions)
Immobilon, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Anti actin, by Merck Group (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using tbs blocking buffer
1
Quantifying Cathelicidin Protein Expression
2
Western Blot Analysis of Protein Expression
3
Protein Expression Analysis in HeLa Cells
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HeLa cells and WT/D210N HeLa cells were transfected with the siRNA control NC (TTCTCCGAACGTGTCACGT), siSETX (AGCAAGAGAUGAAUUGCCA), siSETX2 (GCCAGAUCGUAUACAAUUA), or siTop1 (GACAAGAUCCGGAACCAGU) for 72 h at 20 nM with RNAiMAX (Invitrogen 13778), and siRPA70 (AACACUCUAUCCUCUUUCAUGUU), siRPA32 (GCACCUUCUCAAGCCGAAA), and siRPA32-2(GGAAGUAGGUUUCAUCUAU) for 72 h at 10 nM with RNAiMAX. Protein samples were collected by 2× sample loading buffer and were separated by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane (Bio-Rad 1620115) according to standard protocols (Richard et al. 2021 (link)). Membranes were blocked in 5% milk–PBST blocking buffer for 30 min at room temperature followed by overnight incubation with the primary antibody diluted in protein-free (TBS) blocking buffer (1:1000; Thermo Scientific, 37570), except for GAPDH and H3 (1:10,000). Secondary antibody was added at 1:25,000 for 30 min at room temperature in PBST + 5% milk. Protein bands were detected by chemiluminescence and signals were captured with a ChemiDoc MP imaging system (Bio-Rad). Multiple exposures were performed to ensure signal were in linear range. All protein signals were normalized to GAPDH or H3.
4
Histone Western Blot Analysis
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Histones were separated using SDS-PAGE and transferred to a nitrocellulose membrane using the iBlot Dry Blotting System (Life Technologies) following the manufacturer’s protocol. The membranes were blocked with TBS blocking buffer (Thermo scientific/37543) for 1 hour at room temperature, then incubated in primary antibody at 4°C overnight and secondary antibody for 1 h at room temperature. The following dilutions were used for primary antibody incubation: 1∶50000 H3 (ab1791), 1∶1000 HA (12CA5), 1∶500 tH3 (3008-72).
5
Western Blot Analysis of His-Tagged Proteins
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Proteins were transferred electrophoretically to PVDF membranes (Immobilon®, Merck, Germany) by semi-dry transfer. Membranes were blocked with Protein-Free T20 (TBS) Blocking Buffer (Fisher Scientific UK Ltd., United Kingdom) for 1 h. All subsequent incubations and washes were done in Tris-buffer saline containing 0.1% Tween 80 (TBST). Membranes were incubated with monoclonal Anti-6X His tag® antibody conjugated to HRP (Abcam, United Kingdom) at 1:2000 dilution followed by three washes of 5 min each. The membrane was finally washed with TBS and antibody reactive bands were revealed using ECL Western Blotting Detection Reagent (Geneflow, United Kingdom) and imaged using a Syngene G-Box (Syngene, United Kingdom).
6
Protein Expression Analysis in Neurological Samples
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TZ and PBZ samples (n = 11) were lysed in RIPA buffer containing a protease inhibitor cocktail and PMSF (Fisher Scientific, Illkirch, France) at 4 °C for 30 min. The lysates were clarified by centrifugation at 14,000g at 4 °C for 30 min. Protein concentrations were determined using the Pierce™ BCA protein assay kit (Fisher Scientific) with BSA as the standard, and equal samples of proteins (10 μg/lane) from the samples were resolved on a 7.5% SDS–polyacrylamide gel. The proteins were then electrotransferred onto PVDF membranes. After blocking in TBS blocking buffer (Fisher Scientific) at 4 °C overnight, blots were incubated with the respective primary antibodies [anti-actin (Merk Millipore, Guyancourt, France), anti-neurofilament light polypeptide (NEFL), anti-synapsin 1 (SYN1) and anti-topoisomerase I binding, arginine/serine-rich, E3 ubiquitin protein ligase (TOPORS) (CliniSciences, Nanterre, France)] for 2 h at room temperature. Horseradish peroxidase-conjugated secondary antibodies were then used and visualized with an enhanced chemoluminesence (ECL) reagent and the LAS4000 digital imaging system (Fisher Scientific).
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