Hi all, Anyone can figure out what went wrong in this Western?
What kind of detection method was used for this particular blot?
We added hydrogen peroxide (the secondary antibodies were peroxidase-conjugated)
It would be useful if you could include additional information about what you were trying to do. For example:
1. Is this a Western that you've successfully performed in the past?
2. Are the proteins used of good quality/tested successfully before?
3. Are the antibodies (primary and secondary) a new batch or an old one that were recently used successfully?
4. Do you re-use your antibody dilutions or do you prepare a fresh batch every time?
5. What kind of blocking agent did you use?
1- I will delay answering this question just until I hear your answers ;) (because I was thinking of making this a little simple puzzle).
2- Yes they were. and I have used them successfully before.
3- They were a new batch
4- Not when I carried out this one.
5- I used fat-free milk.
Thanks for asking!
Waiting to start hearing from you :)
What would say went wrong here?
Or do you think something went wrong in the first place?
Based on your answers it seems that this issue is an isolated one. In other words, it could easily have been a small mistake that led to your Western not working. I would not be surprised if you run another Western and that one would work just fine. Just a guess.
Of course ideally you would want to re-make any new solutions that could have caused this problem. For example if you used a new batch of PBS/PBST maybe it would be a good idea to re-make it before trying to run another Western.
Since you mentioned that this is as a puzzle, here is my guess-
Because you only added hydrogen peroxide without any chromogenic substrates during the final detection step??