Lost all of the signals after stripping - WESTERN BLOTTING (Sep/09/2005 )

After I have stripped the filters then one of my filters lost all the signals even the signals of the MW marker. What is wrong? The protocol for stripping is this:


Warm the stripping buffer (62,5mM Tris-HCl pH 6.7 and 2% SDS) to 50C and put the filter in it for 30min at Room Temp (RT). Wash 2X 10min at RT. Block the membrane with 5% non fat dry milk solution.



Any ideas?

Thank you!

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We used a slightly different protocol for stripping blots and rarely had a problem:

Stripping buffer (10 mls)
100mM b-mercapt : 68.5 ul (add in hood)
2% SDS: 2 mls of 10%
62.5 mM Tris-HCl pH 6.7: 624 ul Tris (1M)

Place blot in stripping buffer at 50 degrees for 30 minutes and then wash in blocking buffer (no milk) before re-blocking

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As i said then one of my filters lost all of the signals even the MW marker. I wonder if stripping can do this as a side effect? Yesterday i stripped again and then reprobed with B actin and this time i have got the signals back. Have you experienced this?

Thanks again!

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its weird, but for blotting of some proteins it does happen, for some is fine. me, for exapmle, when looking at my protein, no matter how harsh stripping conditions i use (bme,sds, 70 degrees for 1h), i cannot strip my membrane completelly and still have a strong signal. on the other hand, my friend in the lab usually cannot blot after stripping - she looses the signal. therfore she runs the gels in duplicate.
the other thing - you need to remember to wash off totally the stripping buffer.

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How do you fix the membrane after transfer?

I usually just boil the membrane for 15-30 mins. This greatly improves my signal after stripping it.

ALso, for me it works a 3x 15mins wash with 0.2M NaOH to loose completly my previous signal.

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This is a good thread.

I have tried using this same stripping buffer, 30min, 50c with shaking and I have had two separate NC membranes 100% stripped of all protein after using this protocol... confirmed by ponceau staining after washing away the strip buffer.

Normally I use the 0.2M NaOH for 5min strip protocol detailed in Current Protocols in Immunology, but that has been unsuccessful in stripping phospho-antibodies and their secondary.



This is the first I have heard of fixing a western membrane after protein transfer. Does anyone have any comments on it?

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