Lost all of the signals after stripping, WESTERN BLOTTING

[mountain]

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!

 

[sssddd]

Ok first you need to tell me how big the protein or what proteins u are working with.


just tell me if it's big or small. your procedures seem to be pretty general, don't see any major problems.

2nd tell me how many times have you repeated the experiment

 

[Moonbear]

If it's the same problem he's asked about before that's still unresolved, it would be beta-actin that he's trying to detect after stripping.

https://www.physicsforums.com/showthread.php?t=88152

Welcome to PF sssddd! If you have experience stripping Western's or using them for semi-quantitative analyses, mountain sure could use your advice (we also have another member here struggling with Western's going by the nickname "sotellme"). Both of them, unfortunately, seem to be working in labs lacking sufficient expertise in these methods to help them properly learn and troubleshoot. We're doing the best we can to offer advice here, though it's tough when we can't just watch or show. My own experience with Westerns is pretty limited, so I can't offer them as much advice on troubleshooting as they really need. (They really seem to need new mentors, but that's another issue entirely.)