b'Transfer buffersCommon buffers used for western blotting are the TowbinMethanol in the transfer buffer aids in stripping off SDS buffer system (25 mM Tris-HCl, pH 8.3, 192 mM glycine,from proteins in SDS-PAGE gels, thus increasing their 20% (v/v) methanol) [1] and the CAPS buffer systemability to bind to support membranes. However, methanol (10 mM CAPS, pH 10.5, 10% (v/v) methanol). However, thecan inactivate enzymes required for downstream final choice of transfer buffer may depend on the transferanalyses and can shrink the gel and membrane, which device and will be noted in the device instruction manual. may increase the transfer time of large molecular weight proteins (150 kDa) with poor solubility in methanol. In the In most experiments, SDS should be omitted from theabsence of methanol, though, protein gels may swell in low western transfer buffer because the negative chargeionic strength buffers, and therefore it is recommended imparted to proteins can cause them to pass throughto pre-swell gels for 30 minutes to one hour to prevent the membrane as opposed to binding the membraneband distortion.(also known as blow-through). Typically, residual SDS associated with the proteins in SDS-PAGE gels is sufficientTo increase speed of transfer with semi-dry methods, to effectively carry them out of the gel and onto thehigh ionic strength buffers are the choice. These buffers, membrane support. For proteins that tend to precipitate,when combined with a suitable constant high-current the addition of low concentrations of SDS (0.01%) maypower source (1.5 to 5.0 A), will decrease protein transfer be necessary. It should be noted that adding SDS to thetimes to under 10 minutes.transfer buffer may require optimization of other transfer parameters (e.g., time, current) to prevent proteins fromWe offer several ready-to-use buffers for standard wet, blowing through the membrane. semi-dry, and fast semi-dry blotting systems (Table 10).Table 10. Transfer buffer selection guide.Wet transferProtein sample Gel chemistry Transfer bufferBroad-range MW (6400 kDa) Bolt Bis-Tris Plus Bolt Transfer BufferReduced samples: Bolt AntioxidantBroad-range MW (6400 kDa) NuPAGE Bis-Tris NuPAGE Transfer BufferHigh MW (40500 kDa) NuPAGE Tris-Acetate Reduced samples: NuPAGE AntioxidantBroad-range MW (6400 kDa) Novex Tris-GlycineNovex Tris-Glycine Transfer BufferLow-range MW (2.540 kDa) Novex TricineSemi-dry transferPower Blotter 1-Step Transfer Buffer (5X)Did you knowThe addition of up to 20% methanol or ethanol to a transfer buffer can improve small molecular weight protein transfer.Learn more at thermofisher.com/transferbuffer23'