How should nitrocellulose be treated prior to transferring proteins?
Prewet the nitrocellulose membrane in distilled water or in 1X transfer buffer for 5 min with gentle shaking. This is to prevent any dry spots in the membrane that may interfere with transfer.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
How can I store, strip, and reuse my western blot?
For nitrocellulose or PVDF membrane following Western blot detection using a chemiluminescent or fluorescent substrate system: Following transfer, air dry the membrane and place in an envelope, preferably on top of a supported surface to keep the membrane flat. The blot can be stored indefinitely at -80 degrees C. When ready to reprobe, prewet the PVDF blot with alcohol for a few seconds, followed by a few rinses with pure water to reduce the alcohol concentration. Then proceed as normal with blocking step.
FOR STRIPPING/REPROBING OF MEMBRANES:
Harsh protocol (see NOTE below for modifications)
1) Submerge the membrane in stripping buffer (100 mM BME, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) and incubate at 50 degrees C for 30 min with occasional agitation. If more stringent conditions necessary, incubate at 70 degrees C.
2) Wash 2 x 10 min in TBS-T/PBS-T at room temperature.
3) Block the membrane by immersing in 5% blocking reagent TBS-T or PBS-T for 1 hr at room temperature.
4) Immunodetection
NOTE: Often you don't need such harsh conditions to remove antibodies from their proteins. The stringency of one or several of the variables can be decreased: lower the temperature, decrease the time, less BME, less SDS, etc. An especially mild but still often effective stripping protocol is lower pH incubation. Example: pH 2.0 Tris 50-100 mM, 30-60 min incubation (you may do two incubations if you wish). Then rinse and block as usual. If you do not wish to re-use the membrane immediately after stripping, you can store the membrane in plastic wrap (wet, you do not want it to dry out). Another simple, mild stripping buffer is 0.1 M glycine•HCl (pH 2.5-3.0), incubation 30 min to 2 hrs room temperature or 37 degrees C, depending on the antibody.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
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.
