After transferring onto a PVDF membrane, what is the best way to store the membrane for future probing?
We recommend air drying the PVDF membrane and placing it in an envelope, preferably on top of a supported surface to keep the membrane flat. It can be stored indefinitely at ≤80 degrees C. Right before probing, we recommend re-wetting the membrane with alcohol for a few seconds, followed by a few rinses with pure water to reduce the alcohol concentration. Then proceed as normal with the blocking step.
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Which transfer buffers are recommended for peptide (N-terminal) sequencing?
Use non-glycine based buffers such as the NuPAGE Invitrogen Transfer buffer, CAPS, or 1/2X TBE transfer buffer.
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What parameters can affect Western blotting transfer results?
SDS:
-Mobility out of the gel: increases mobility as SDS imparts negative charge to a protein and maintains it in a soluble state.
-Binding to membrane: reduces binding to nitrocellulose due to decreased hydrophobicity of the protein.
-Detection: may affect antigenicity of some proteins.
Alcohol in transfer buffer (i.e., methanol up to 20%):
-Mobility out of the gel: decreased; reduces pore size of gel.
-Binding to membrane: improves binding to nitrocellulose; removes SDS from proteins resulting in improved hydrophobic interactions.
Field strength (V/cm):
-Mobility out of the gel: field strength is the driving force of elution; if too low, sample is not completely transferred out of gel.
-Binding to membrane: if too high, sample passes through membrane without binding.
Transfer buffer:
-Mobility out of the gel: decreased if high conductivity and low resistance limit V/cm due to excessive heat generation; decreased if buffer pH is close to pI of native protein.
Membrane:
-Detection: PVDF and nylon require more stringent blocking conditions than nitrocellulose.
Gel:
-Mobility out of the gel: increases with thinner gels or larger gel pore size.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
How can I reduce background bands in my Western blot?
Optimize the concentration of primary and secondary antibodies. In some cases, increasing the concentration of blocking agent (BSA or non-fat dry milk) or usiing an alternative blocking solution such as Starting Block or SuperBlock may reduce background signal. After incubation with the primary antibody, wash at least 2 times with TBST (include 0.5 M NaCl in one or more of the wash steps). Avoid Nonidet P40 or Triton X-100 in buffers because protein detection is decreased when these detergents are used.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
How can I increase the sensitivity of my Western blot?
Consider transferring to a different membrane or using a different detection method. We have observed increased sensitivity when using PVDF membranes in place of nitrocellulose. On PVDF membranes, using as little as 1 µg of total rat brain protein, PKC can be detected with alkaline phosphatase-mediated chromogenic detection in some cases using affinity-purified antibodies at a concentration of 0.5 µg/mL. Detection sensitivity can also be increased by using chemiluminescent detection, especially when using a SuperSignal West enhanced chemiluminescence subtrate (https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-assays-analysis/western-blotting/detect-proteins-western-blot/western-blot-detection-reagents/detection-technologies-western-blotting/chemiluminescent-western-blot-detection/supersignal-chemiluminescent-substrates.html) such as SuperSignal West Pico PLUS, SuperSignal West Dura, or SuperSignal West Femto. The secondary antibody should be used as recommend by the manufacturer and optimized as needed.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
