小中大
这是我从amersham pharmacia 的说明书上照着敲下来的。敲到最后一行，发现指向第14页，一翻还挺多，就不敲了。如果需要就到他们的网站上去载吧。
Hybond-PVDF membrane (optimized for protein transfer)
p22-23
Protocols for reprobing membranes
Following eCL or ECl Plus detection it is possible to reprobe the membrane several times to either clarify or confirm results or when small or valuable samples are being analyxed. Sequential reprobing of membranes with a variety of antibodies is possible. The blots must be stored wet wrapped in Saran Wrap at 2-8 degree after each immunodetection.
Protocol One
This procedure is suitable for sequential reprobings using primary and secondary antibodies raised in differernt species using ECL systems or where different immunodetection systems have been used.
Protocol
1. Reapply prepared ECL detection reagents to the blots and re-expose to a sheet of autoradiography film.
2.If a signal is detected incubate the blot in prepared ECL detection reagents for 30 min. Repeat stepI. If nosiganl is detected proceed to step3.
3.Perform immunodetection using a primary antibody raised in a different species to the first. Secondary antibodies must demonstrate no cross reactivity.
Protocol Two--stripping and reporbing membranes
The complete removal of primary and secondary antibodies from membranes is possible following the method outlined below The membrane may be stripped of bound antibodies and reporbed several times. this procedure amy anot be suitable for enzyme substrates where insluble reaction products are deposited on the membrane. membranes should be sored wet wraaped in Saran Wrap at 2-8 degree after each immunodection.
In excess of 50% of some target proteins can be lost when performing experiments where blots are stripped and reprobed. It is therefore important to consider which antigen is present in least abundance and probe for this first.
Protoco
1.Submergethe membrane in stripping buffer(100mM beta-mercaptoethanol, 2%(w/v) sodium dodecyl sulphate, 62.5 mm tris-HCl pH6.7) and incubate at 60 degree for 30min with occasional agitation.
2. wash the membrane for 2x10 min in TBS-T or PBS-T at room temperature suing large volumes of wash buffer.
3.Block the membrane by immersing in 5% (w/v) blocking reagent in TBS-T or PBS-T for 1 hour at room temperature.
4. Perform immunodetection as descri ed on P14.
