If Invitrogen™ pre-cut membrane/filter sandwiches are used, at least 2 extra filter papers (0.4 mm/filter in thickness) on each side of the gel (or membrane) are required. NP0006), mix the following: 10 mL of 20X transfer buffer, 10 mL of MeOH, 100 µL of Antioxidant, 80 mL of DI H2O.įilter papers: The transfer buffer-soaked filter papers of the sandwich are the only reservoir in the Semi-Dry Transfer Cell. If you need to prepare 100 mL of the working buffer from the NuPAGE™ 20X Transfer Buffer (Cat. Working transfer buffer: 10% methanol, 1:1,000 Antioxidant in 2X NuPAGE™ transfer buffer (Bis-Tris 50 mM and Bicine 50 mM). NuPAGE™ transfer buffer can be used for transfer of Tris-Glycine gels. Here is the transfer protocol optimized for the Bio-Rad Semi-Dry Transfer Unit. Note: For transfer of large proteins (>100 kDa), pre-equilibrate the gel in 2X NuPAGE™ Transfer Buffer (without methanol) containing 0.02-0.04% SDS for 10 min before assembling the sandwich. Perform the transfer at 20 V (constant) for 30–60 minutes. If you are not using the Novex™ pre-cut membrane/filter sandwiches, use two thick filter papers.Įquilibrate the gel in the transfer buffer (100 mL for Midi gels and 50 mL for Mini gels) for 10 minutes, on an orbital shaker, to remove salts that may increase conductivity and heat during transfer.Īssemble the gel/membrane/filter paper sandwich on top of the anode plate as follows: If you are using Novex™ pre-cut membrane/filter sandwiches, use three filter papers (0.4 mm/filter in thickness) on each side of the gel or membrane. Soak the filter paper and transfer membrane in the transfer buffer. If you are blotting large proteins, please see the Note below. NuPAGE™ Antioxidant (for reduced sample) 0.1 mL Prepare 100 mL of 2X NuPAGE™ Transfer Buffer from 20X NuPAGE™ Transfer Buffer as follows: If you decide to use the Novex™ Semi-Dry Blotter for NuPAGE™ -Tris Gels, use the protocol provided below to ensure efficient transfer of proteins. NuPAGE™ Bis-Tris Gels do not transfer efficiently using the Novex™ Semi-Dry Blotter as compared to blotting with XCell II™ Blot Module. Floating the gel over the filter paper avoids the need to handle the gel and prevents the gel from getting stuck onto the filter paper before it is in its proper position. When the gel is in the correct position, lift up on the filter paper so the gel attaches to it. While the gel is floating in the equilibration solution, submerge the filter paper underneath the gel. Tip for handling the IEF gel: The 5% polyacrylamide IEF gels tend to be sticky. This is opposite from the typical western blot with SDS, where the negatively charged protein will migrate toward the anode (+) side during the transfer. Assemble the gel/membrane sandwich in reverse order so that the membrane is in contact with the side of the gel facing towards the cathode (–). Chill the 0.7% acetic acid that will be used as the transfer buffer. The following protocol prevents the gel from hydrolyzing and is especially effective for basic proteins because of the low pH of the transfer buffer.Īfter the run, equilibrate the gel in 0.7% acetic acid (0.7% acetic acid in water, pH 3.0) for 10 minutes. The IEF gels are 5% polyacrylamide and transferring them in a basic buffer leads to substantial hydrolysis and damage to the gel. For IEF gels, we recommend using an acetic acid transfer buffer.
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