低荧光 PVDF 转印膜，0.2 μm 7 x 8.4 cm

以下是可能原因和解决方案：

- 转印前对膜的处理不当：应确保使用甲醇或乙醇等极性有机溶剂润湿膜。
- 凝胶与膜的接触不良：应确保用转膜缓冲液浸透滤纸和海绵垫，小心除去组装膜三明治时的所有气泡。凝胶/膜三明治必须牢固装在两部分印迹模块之间。可尝试多加一个海绵垫或将失去弹性的海绵垫换成新的。
- 过度挤压凝胶：过度挤压的一个明显表现是凝胶过于扁平。在三明治被过度挤压的情况下，应适当移除海绵垫，降低对凝胶和膜施加的过多压力然后关闭转印仪。

可以，我们的PVDF和硝化纤维素膜均可用于Li-COR仪器。

我们的PVDF膜可以耐受乙腈，但硝化纤维素膜不能耐受。

PVDF膜需要更为严格的封闭步骤。这可通过将封闭剂的浓度增加2-5倍、延长封闭时间并在37℃封闭来实现。封闭剂与无蛋白质结合的位置结合，可防止背景染色，并封闭与膜结合的蛋白质，以减少其与一抗发生非特异性相互作。封闭剂可使用脱脂奶粉、BSA和酪蛋白。

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.

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