Consider extending transfer time or increasing voltage.Ĭonversely, small proteins migrate quickly and may be pushed through the membrane. Large proteins migrate more slowly out of the gel. Consider additional cooling or limiting transfer time.Ĭurrent increases when temperature increases. Limit transfer time to prevent small proteins from "blowing through" membrane.Ĭurrent increases when volume increases. Proteins move quickly through gels with low acrylamide percentages. Higher acrylamide percentages slow protein migration out of the gel. Consider additional cooling or limiting transfer time. See Our Western Blotting Transfer Membranes »Īddition of SDS or changes in ion concentration due to addition of acid or base, changes the resistance of the transfer system and therefore current and results in changes in current and voltage readings as well as transfer efficiency.Ĭurrent increases slightly as the number of gels increases. 0.2 μm pore size membranes are most suitable for transfer of low-molecular-weight (0.45 μm pore size membranes are recommended for most analytical blotting experiments.Membranes are commonly available in two pore sizes: This low autofluorescence allows longer exposure times without increasing background fluorescence levels, allowing detection of faint signals. Low-fluorescence formulations of PVDF are available and combine the advantages of PVDF with low autofluorescence across a wide range of excitation and emission wavelengths. PVDF membrane has a strong protein binding capacity (about twice that of nitrocellulose) and will not crack nor tear in common handling, so it can withstand repeated stripping and reprobing. This extra support gives the membrane increased strength and resilience to withstand reprobing and autoclaving (121℃) while maintaining the ease of wetting of nitrocellulose. Supported nitrocellulose is an inert support structure with nitrocellulose applied to it. Unsupported nitrocellulose is innately fragile and are not recommended for stripping and reprobing. Nitrocellulose membranes are easily wetted in water or transfer buffer, and it is compatible with a wide range of protein detection systems. Nitrocellulose and Supported Nitrocellulose But alcohol may cause a reduction in the gel pore size, precipitation of some proteins, and some basic proteins to become positively charged or neutral.Ĭonsiderations for Selecting Nitrocellulose or PVDF to use for Western Blots However, SDS in the transfer buffer decreases the binding efficiency of protein to nitrocellulose membrane PVDF membrane can be substituted if desired.Īlcohol, on the other hand, removes the SDS from SDS-protein complexes and improves the binding of protein to nitrocellulose membrane. SDS promotes elution of the protein from the gel and in cases where certain proteins are difficult to elute from the gel, SDS may be added to the transfer buffer. SDS and alcohol play opposing roles in a transfer. Use acid-gel transfer protocol (membrane toward cathode) Tank blotting recommended temperature regulation may be needed to maintain activity Tank blotting recommended needs high-capacity, small pore-size membrane pH of buffer may be criticalĭepends on pH of gel buffer and pI of protein of interest Towbin with or without SDS, CAPS, carbonate, Bjerrum Schafer-Nielsen Transfer conditions must be optimized for proteins of different molecular weights, e.g., to prevent under-transfer (incomplete transfer of proteins out of the gel) or over-transfer (loss of proteins passing completely through the membrane). Both the voltage and the distance between the electrodes then play a major role in governing the rate of elution of the proteins from the gel. ![]() The electric field strength (measured in V/cm) that is generated between the electrodes is the driving force for transfer. Voltage is applied between the electrodes and proteins migrate to the membrane following the current that is generated by the applied voltage across the electrodes. During this process, the membrane and gel are placed together, with filter paper between two electrodes. ![]() The most common method of transfer in western blotting is electrophoretic transfer, where an electric field is used to elute proteins from gels and transfer them to membranes. Gel and Membrane Setup for Electrophoretic Transfer
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