![]() ![]() If you are using 19:1, you should try 29:1. For example, generally protein gels are around 29:1 acrylamide-to-bis-acrylamide.Using 5% C (with bis-acrylamide as the crosslinker) produces the smallest pore size Reduce %T (total monomer) or %C (crosslinker) to increase gel pore size and increase transfer efficiency.Gel percentage too high, decreasing transfer efficiency Reduce or eliminate the alcohol in the transfer buffer.SDS can increase transfer efficiency but it can also reduce binding efficiency to nitrocellulose and affect reactivity of some proteins with antibodies A protein near its isoelectric point (pI) will transfer poorly (buffer pH should be 2 pH units higher or lower than the pI of the protein of interest for optimal transfer efficiency) Use a more basic or acidic transfer buffer to increase protein mobility.Incorrect charge-to-mass ratio (native transfers) Check the polarity of the connections to the power supply.Check the assembly of the transfer cell, and ensure that the cassette is inserted into the tank in the correct orientation.Sandwich assembly for semi-dry blotting: Bulletin 6214.Sandwich assembly for tank blotting: Bulletin 6213.Check that the gel/membrane sandwich has been assembled in the correct order.Protein transferred away from the membrane If it does not match your needs, get a power supply with a higher current capacity such as the PowerPac™ HC Power Supply Check the output capacity of the power supply to ensure the voltage and current output match the needs of the blotting instrument.Power supply circuit inoperative or an inappropriate power supply used Use PVDF or 0.2 µm nitrocellulose (smaller pore size).Place an additional membrane in the gel sandwich to detect any low-MW proteins that are transferred through the membrane.Decrease the voltage, especially if using a high-intensity transfer device, e.g., Semi-Dry and Rapid Blotting Systems such as the Trans-Blot SD or Trans-Blot Turbo Systems.Proteins Low-MW proteins may be transferred completely through the membrane.Power conditions too high or transfer time too long If an incorrect power supply is used, it may not reach the set voltage if the current of the power supply is at its maximum limit Use a power supply with a high current limit such as the PowerPac™ HC Power Supply.Use high-intensity blotting devices, e.g., Semi-Dry and Rapid Blotting Systems such as the Trans-Blot ® SD or Trans-Blot ® Turbo™ Systems.See the power guidelines for specific applications in Table 4.1 of the Protein Blotting Guide ( Bulletin 2895) Check the current at the beginning of the run it may be too low for a particular voltage setting, indicating incorrect buffer composition.Increase the transfer time (thicker gels require longer transfer times).Power conditions inadequate or transfer time too short ![]() Optimize transfer buffers for methanol and SDS concentrations.Optimize transfer conditions for target protein size.Confirm transfer with Bio-Rad’s Stain-Free Imaging Technology.Confirm transfer with Ponceau S staining.(see below for more details on transfer power conditions, gel electrophoresis, and buffer preparation see also Western Blotting > Transfer Conditions) Optimize sample loading see Determining the Appropriate Sample Load for Western Blots Protocol.Fractionate or concentrate the sample using one or more of these techniques:.Check concentration of protein samples (e.g., using Bradford or Lowry protein assays).(see also Protein Band Appearance > No Bands)Īll bands appear very faint, or you see no signal with chemiluminescence or fluorescence detection.įor more hints on how to improve transfer and binding, see also Troubleshooting: Transfer in the Protein Blotting Guide Make sure the membrane is fully immersed and agitated throughout incubations.Not enough solution used during incubation and/or washing Transfer tank must contain sufficient buffer to entirely cover blot area Completely fill transfer tank with buffer.Ensure that warm membranes are not allowed to dry after transfer by shortening handling time High-intensity or rapid transfer methods generate heat that may cause the membrane to dry while it is handled.A completely wet PVDF membrane has a gray, translucent appearance When using a hydrophobic PVDF membrane, it must be completely soaked in methanol prior to equilibration in aqueous transfer buffer.Ensure that membranes are uniformly wet before transfer.Membrane not uniformly wet before transfer Some sections of the blot appear to have less protein on them. ![]()
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