b'Washing the membrane lose their properties and potency as solubilizing agents. We offer several high-purity, low peroxidecontaining Like other immunoassay procedures, western blottingdetergents that are packaged under nitrogen gas in clear consists of a series of incubations with different antibodiesglass ampules. These Thermo Scientific Surfact-Amps separated by wash steps. Washing steps are necessary todetergent solutions provide superior convenience, quality, remove unbound or weakly bound reagents and to reduceand consistency for all detergent applications. Commonly background, increasing the signal-to-noise ratio. Insufficientused detergents for western blotting include Tween 20 and washing produces high background, while excessiveNP-40 detergent solutions.washing may result in decreased sensitivity caused by elution of the antibody or target protein from the blot. AsBupH Phosphate Buffered Saline Packs (PBS)with other steps in performing a western blot, a variety ofEach pack of Thermo Scientific BupH Phosphate buffers may be used (Table 15). Occasionally, washing isBuffered Saline yields 500 mL of 0.1 M phosphate, performed in a physiological buffer such as Tris-buffered0.15 M sodium chloride, pH 7.0, when dissolved in saline (TBS) or phosphate-buffered saline (PBS) without500 mL deionized water (20 L total).any additives. More commonly, a detergent such as 0.05% Tween 20 detergent is added to the buffer to help removeBupH Tris Buffered Saline (TBS)nonspecifically bound material. Another common techniqueEach pack of Thermo Scientific BupH Tris Buffered Saline is to use a dilute solution of the blocking buffer along withyields 500 mL of 25 mM Tris, 0.15 M sodium chloride, some added detergent to help minimize background.pH 7.2, when dissolved in 500 mL deionized water (10 packs make 5 L total; 40 packs make 20 L total).Although detergents are available from several commercial sources and used routinely in many researchTween 20 Surfact-Amps Detergent Solution laboratories, the importance of detergent purity andThermo Scientific Tween 20 Surfact-Amps Detergent stability is not widely appreciated. Detergents oftenSolution is a highly purified Tween 20 detergent stabilized contain trace impurities from their manufacture. Some ofas a 10% solution and packaged under nitrogen in glass these impurities, especially peroxides that are found inampules or nonleaching, high-density polyethylene most nonionic detergents, will destroy protein activity.(HDPE) bottles, ensuring its stability and eliminating the In addition, several types of detergents oxidize readilyaccumulation of peroxides and degradation products.when exposed to the air or UV light, causing them to Table 15. Wash buffer selection guide.TBS PBS TBST PBSTFormulation Both dry and liquid: Dry:25 mM Tris10 mM sodium phosphate 25 mM Tris100 mM sodium phosphate150 mM NaCl150 mM NaCl 150 mM NaCl 150 mM NaCl0.05% Tween 20 detergent0.05% Tween 20 detergent pH 7.2pH 7.2 pH 7.5pH 7.5Liquid: 10 mM sodium phosphate 150 mM NaClpH 7.5 Dry blend Cat. No. 28376 (40 packs)Cat. No. 28372 (40 packs) NA NACat. No. 28379 (10 packs)Liquid concentrate Cat. No. 28358 (500 mL) Cat. No. 28348 (500 mL) Cat. No. 28360 (500 mL) Cat. No. 28352 (500 mL)ApplicationELISA, western blotting, ELISA, western blotting, and ELISA, western blotting, ELISA, western blotting, and other immunoassays other immunoassays and other immunoassays and other immunoassays Use with HRP, AP, or Crosslinking, biotinylation, and Use with HRP or AP Use with HRP detection fluorescent detectionfluorescent labeling reactionsdetection systems; cansystems; can be used systems requiring an amine-free buffer be used in fluorescentin fluorescent detectionUse with HRP or fluorescentdetection systems aftersystems after blocking stepdetection systems blocking stepLearn more at thermofisher.com/westernbuffers41'