Lost all of the signals after stripping, WESTERN BLOTTING

In summary, the conversation discusses stripping filters and the loss of signals, and the protocol for stripping is outlined. The speaker also asks for ideas and clarification on the proteins they are working with. Another member joins the conversation and offers advice on Westerns and suggests seeking help from more experienced mentors.
  • #1
mountain
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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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  • #2
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
 
  • #3
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.)
 

What is western blotting and why is it used?

Western blotting is a laboratory technique that is used to detect specific proteins in a sample. It involves separating and transferring proteins from a gel to a membrane, and then using antibodies to bind to the specific protein of interest. This technique is commonly used in research and diagnostic settings to determine the presence, quantity, and size of a particular protein.

What does it mean when all of the signals are lost after stripping in western blotting?

After performing a western blot, the membrane is often stripped to remove the bound antibodies and re-probed with a different antibody. Losing all of the signals after stripping means that no proteins were detected by the second antibody, indicating that the protein of interest is not present in the sample.

What are some reasons for losing all of the signals after stripping in western blotting?

There are several possible reasons for losing all of the signals after stripping in western blotting. These include not enough protein being loaded onto the gel, improper transfer of proteins to the membrane, or using an antibody that is not specific for the protein of interest. Other factors like poor antibody quality, incorrect dilution of antibodies, or inadequate blocking of the membrane can also lead to loss of signals.

Can western blotting be used to detect multiple proteins at once?

Yes, western blotting can be used to detect multiple proteins at once. This can be achieved by using different types of antibodies that are specific to different proteins, or by performing a technique called multiplexing where multiple antibodies are labeled with different fluorescent dyes and used simultaneously on the same membrane.

What are some troubleshooting tips for when signals are lost after stripping in western blotting?

If all signals are lost after stripping in western blotting, some troubleshooting steps to consider include optimizing the amount of protein loaded on the gel, ensuring proper transfer of proteins to the membrane, using high quality and specific antibodies, and performing a thorough blocking step. It may also be helpful to try different stripping methods or conditions, as well as adjusting the detection method or exposure time.

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