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My Account. Browse Catalog. Life Science Research Back. Life Science Research Explore all. Bio-Rad Products Explore all. Support Explore all. Clinical Diagnostics Explore all. Process Separations Explore all. Consider additional cooling or limiting transfer time. Higher acrylamide percentages slow protein migration out of the gel. Consider extending transfer time. Proteins move quickly through gels with low acrylamide percentages. Limit transfer time to prevent small proteins from "blowing through" membrane.
Current increases when volume increases. Consider additional cooling. Large proteins migrate more slowly out of the gel. Consider extending transfer time or increasing voltage. Conversely, small proteins migrate quickly and may be pushed through the membrane. Consider limiting transfer time or voltage. For best transfer results, use the highest electric field strength possible within the heat dissipation capabilities of the system.
During transfer, the buffer warms as a result of the power dissipated through the system, and its resistance drops. Such heating and changes to resistance may lead to inconsistent field strength and transfer, and may cause the transfer buffer to lose its buffering capacity, or may cause the gel to melt and stick to the membrane. In general, transfer from thicker or higher percentage gels, or transfer of high-molecular-weight proteins require high electric fields or longer transfer times.
Complete protein elution from the gel can be confirmed by post-staining the gel with a total protein stain. Conversely, transfer from thin, low-percentage gels or of proteins of low molecular weight require lower field strengths and shorter transfer times. This over-transfer can be detected by placing a second membrane behind the first and staining this membrane with a total protein stain or by imaging the backup membrane using a stain-free imaging system.
If the voltage is held constant throughout a transfer, field strength remains constant, providing the most efficient transfer possible for tank blotting methods. However, the current in tank transfer systems increases as the resistance drops due to heating; in most semi-dry systems, current drops as a result of buffer depletion.
Therefore, the overall power increases during transfer, and there is an increased risk of heating. Use of the cooling elements available with the various tank blotting systems helps prevent problems with heating.
If the current is held constant during a run, a decrease in resistance results in a decrease in voltage and power over time and therefore lower risk of heating. However, proteins are transferred more slowly due to decreased field strength. Uniform protein transfer requires complete contact between the components of the transfer stack, especially between the gel and membrane.
Tip: Gradient gels are best rolled up-down, rather than side-to-side. Side-to-side rolling causes gradient gels to warp and wrinkle. When performing traditional wet tank transfers and traditional semi-dry transfers, optimal results are obtained when the electrophoresis buffer saturating the gel is replaced with transfer buffer.
Contaminating buffer salts increase the conductivity of the transfer buffer resulting in excess heat during the transfer. Rinse gels briefly in water then equilibrate the gel for 15 minutes and the membrane for at least 5 minutes in transfer buffer. Transfer requires that the membrane stays wet in transfer buffer both before and after transfer. Heat generated during a transfer can dry out membranes once removed from the transfer stack.
This is especially important with PVDF membranes since they are hydrophobic. Work quickly, and have a dish of buffer ready to immerse your membrane into in order to avoid drying the membrane. If they do dry, nitrocellulose can simply be re-wet in buffer while PVDF membranes will require re-activation in alcohol.
Details on blotting technology, methods, products, tips, techniques, and troubleshooting guidelines. Our self-help troubleshooting guide covers solutions to many common and not-so-common western blotting issues and helps your blots look their best.
Learn more about the relative transfer efficiencies of tank, semi-dry, and rapid blotting systems for western blotting with large proteins. Choose the right blotting membrane material, porosity, and form factor for your western blotting experiments. Get Your Free Guide. Join us for an in-depth experience learning about western blotting principles and techniques. This five-part webinar series is for anyone who has ever asked how to perform a western blot and would like to have a detailed understanding of western blotting principles at each step of the workflow.
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Save Preferences. About the Program Explore all. Corporate Explore all. About Bio-Rad Back. About Bio-Rad Explore all. Investor Relations Explore all. Description Specifications Ordering Accessories Documents. Protein Blotting Detection and Imaging. Nitrocellulose Membrane, 0. Specifications Maximum gel size W x L , cm. Buffer requirement, ml. Gel capacity. Recommended power supply. Dimensions W x L x H , cm. Ordering items Use the filters below to refine results! Semi-dry electrophoretic transfer cell, includes agarose gel support frame, extra thick blot paper in 4 sheet sizes.
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