Nitrocellulose/Filter Paper Sandwiches, 0.45 μm, 8.6 x 13.5 cm - FAQs

查看更多产品信息 Nitrocellulose/Filter Paper Sandwiches, 0.45 μm, 8.6 x 13.5 cm - FAQs (LC2006)

2 个常见问题解答

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.