Hello! I am currently running a western blot using human AD tissue samples. I prepared these samples over a year and a half ago. I am having trouble standardizing these proteins with Beta Actin. I have ruled out other parameters that could be causing the problem. Therefore, I am wondering if my tissue samples are no longer reliable. In addition to this, I noticed that when i thaw my samples, there is a good amount of precipitation. I just vortex and spin down to mix the samples thoroughly.
For my results, I keep seeing bands stuck in the wells and multiple faded bands. Of course when I first ran these proteins, the beta actin signal was clean and neat.
If someone could please elaborate on what could be causing this. In addition, I would appreciate if you could provide how long samples last and if there is a way to troubleshoot this.
Thank you!
P.S. Samples have been stored in -20 C fridge.
Hi! For long storage periods, I usually store at -80°C, and they work even after more than a year. As far as I know, it is not advisable to keep protein lysates at -20°C for more than 3 months.
For long term storage even at -20°C, I use glycerol to maintain the solubility and compactness of the protein. I get clear bands even after 6 months of storage.
You detected precipitation after storage, which I think might be resulted from the loss of proteins stability during storage. I hope it will be helpful for you.
- Badges
- Science topic