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View additional product information for Invitrolon™ PVDF/Filter Paper Sandwiches, 0.45 μm, 8.3 x 7.3 cm (for mini gels) - FAQs (LC2005)
11 product FAQs found
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.
Drying the PVDF membrane reduces the background staining that can occur with wet membranes. A dry PVDF membrane is very hydrophobic and doesn't wet well, but the areas where proteins are bound are more easily saturated and stainable.
This will not work with nitrocellulose membranes, and will only work with PVDF membranes stained for a brief period; staining beyond the recommended time will only increase the background and reduce the detectability.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
After staining with SimplyBlue SafeStain, use deionized water for the less strongly retained protein bands on the PVDF membrane.
Increasing methanol or ethanol concentrations up to 70% should destain any remaining bands. You can leave the membrane in the destain indefinitely.
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We find that including ethanol in the transfer buffer is just as effective as including methanol. You may use either of these alcohols at an equivalent concentration.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
PVDF membranes require more stringent blocking steps. This can be achieved by increasing the concentration of the blocking reagent 2 to 5 fold, increasing the blocking time, and performing the procedure at 37 degrees C. Blocking agents bind to unoccupied sites to prevent background staining and also to membrane-bound proteins, thus reducing non-specific interactions with the primary antibody. We offer WesternBreeze Immunodetection kits with blocking reagents and Pierce Fast Western Blot kits that have been pre-optimized to give low-background blots. Other examples of blocking agents are nonfat dry milk, BSA, and casein, Starting Block, and SuperBlock.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
There are three main reasons why this may occur.
1) The membrane may not be properly treated prior to transfer. Make sure that the membrane is pre-wetted with a polar organic solvent such as methanol or ethanol. Failure to do so will prevent proteins from forming hydrophobic interactions with the membrane.
2) There may be poor gel-to-membrane contact. Ensure that the filter paper and blotting pads are well saturated with transfer buffer, taking care to remove any bubbles during the assembly of the membrane sandwich. The gel/membrane sandwich must fit securely in the two halves of the blot module. Try adding another pad or replace any pads that have lost their resiliency with new ones.
3) Overcompression of the gel: a good indication of overcompression is if the gel has been excessively flattened. In the event that the sandwich is overcompressed, remove enough pads so that the blotter can be closed without exerting excess pressure on the gel and membrane.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
The most probable cause of the swirled artifact is the way the membrane is moved around in the staining solution. Using an orbital shaker can cause this effect. To eliminate these background patterns, we recommend switching to a rocker type shaker or combination of rocker and reciprocal motions to ensure even sloshing of the staining solution over the membrane.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Here are possible causes and solutions:
- The membrane may not be properly treated prior to transfer: Make sure that the membrane is pre-wetted with a polar organic solvent such as methanol or ethanol.
- There may be poor gel to membrane contact: Ensure that the filter paper and blotting pads are well saturated with transfer buffer, taking care to remove any bubbles during the assembly of the membrane sandwich. The gel/membrane sandwich must fit securely in the two halves of the blot module. Try adding another pad or replace any pads that have lost their resiliency with fresh ones.
- Over-compression of the gel: A good indication of over-compression is if the gel has been excessively flattened. In the event that the sandwich is over-compressed, remove enough pads so that the blotter can be closed without exerting excess pressure on the gel and the membrane.
Find additional tips, troubleshooting help, and resources within our Protein Electrophoresis and Western Blotting Support Center.
Yes, both our PVDF and nitrocellulose membranes are compatible with the Li-COR instrument.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
Our PVDF can tolerate acetonitrile but our nitrocellulose cannot.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.
PVDF membranes require more stringent blocking steps. This can be achieved by increasing the concentration of the blocking agent 2-5 fold, increasing the blocking time, and performing the procedure at 37 degrees C. Blocking agents bind to unoccupied sites to prevent background staining and also to membrane-bound proteins, reducing non-specific interactions with the primary antibody. Examples of blocking agents are nonfat dry milk, BSA, and Casein.
Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.