Mild stripping buffer

Manufactured by Thermo Fisher Scientific

Mild stripping buffer is a laboratory reagent used to gently remove bound proteins or other molecules from a surface or membrane during various analytical procedures. It is designed to preserve the integrity of the sample or target analyte while effectively removing unwanted components. The core function of this buffer is to facilitate the stripping or disassociation of bound materials without causing significant damage to the sample.

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Lab products found in correlation

Pvdf membrane, by Thermo Fisher Scientific (2 mentions) Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions) Skim milk, by Merck Group (1 mentions) Goat pab to rb igg hrp, by Abcam (1 mentions) Odyssey infrared imaging system, by LI COR (1 mentions) Ecl western blotting substrate, by Bio-Rad (1 mentions) Anti β actin antibody, by Abcam (1 mentions) Complete mini edta free, by Roche (1 mentions) Coomassie protein assay reagent, by Merck Group (1 mentions) Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions) Western ecl substrate, by Bio-Rad (1 mentions) Chemidoc xrs system, by Bio-Rad (1 mentions) Phostop, by Roche (1 mentions)

2 protocols using mild stripping buffer

CCR5 Protein Expression Analysis

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Cells were washed with ice-cold PBS and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo-Fisher Scientific). The total protein concentration in the cell lysates was measured using the bicinchoninic acid assay (Thermo-Fisher Scientific). Subsequently, 30 µg of total protein was separated by SDS-PAGE and transferred onto a PVDF membrane (Thermo-Fisher Scientific). Blocking was carried out using 5% skim milk (Sigma) in 0.05% PBST for 24 h, followed by overnight incubation with the primary antibody (anti-CCR5 antibody; CUSABIO TECHNOLOGY, cat#CSB-PA006994). After washing the blots five times with 0.05% PBST, they were probed with the secondary antibody (Goat pAb to Rb IgG (HRP); AbCam, cat#ab205718) for 1 h and subsequently detected using ECL Western Blotting Substrate (Bio-Rad, cat#1705060) and scanned using the Odyssey InfraRed Imaging System (LI-COR BioSciences, Lincoln, NE). The blots were then stripped with mild stripping buffer (Thermo-Fisher Scientific), re-blocked, and re-probed with anti-β-Actin antibody (Abcam, cat#ab8227).

Khamaikawin W., Saisawang C., Tassaneetrithep B., Bhukhai K., Phanthong P., Borwornpinyo S., Phuphuakrat A., Pasomsub E., Chaisavaneeyakorn S., Anurathapan U., Apiwattanakul N, & Hongeng S. (2024). CRISPR/Cas9 genome editing of CCR5 combined with C46 HIV-1 fusion inhibitor for cellular resistant to R5 and X4 tropic HIV-1. Scientific Reports, 14, 10852.

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Western Blot Analysis of Protein Samples

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Cell lysates were prepared using RIPA buffer (Boston BioProducts) containing protease cocktail inhibitor (cOmplete Mini EDTA- free, Roche) and phosphatase cocktail inhibitor (Phostop, Roche). Protein concentration was determined by coomassie protein assay reagent (Sigma-Aldrich). Twenty μg of total protein was resolved by a NuPAGE 4-12% Bis-Tris gel (Life Technologies), and transferred to PVDF membrane (Thermo Fisher Scientific). After blocking with 5% non-fat milk (M-0841, LabScientific), the membrane was incubated with a primary antibody overnight at 4 °C. Subsequently, the membrane was incubated with a secondary antibody for 90 minutes at room temperature. Western ECL substrate (1705061, Bio-Rad) was used to develop the immunoblot. The picture was captured and analyzed by Image Lab (Version 5.2.1) using ChemiDoc™ XRS+ System (Bio-Rad). When probing for multiple targets, stripping and re-probing a single membrane were applied. The membrane was stripped using mild stripping buffer (46430, Thermo Scientific) for 5 minutes at room temperature in order to avoid stripping out the sample protein on the membrane. The results were quantified by plotting the intensity of the band. β-actin was used as the loading control.

Pan W., Nagpal K., Suárez-Fueyo A., Ferretti A., Yoshida N., Tsokos M.G, & Tsokos G.C. (2021). The regulatory subunit PPP2R2A of PP2A enhances Th1 and Th17 differentiation through activation of the GEF-H1/RhoA/ROCK signaling pathway. Journal of immunology (Baltimore, Md. : 1950), 206(8), 1719-1728.

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