I have 2 month old lysate. I want to do western blot analysis for HDAc 1-7 using this lysate. Can I use this lysate? In other words I want to ask about protein stability of HDAcs in the lysate, whether HDACs remain stable for 2-3 months in the lysate or not? The cell line I am using for lysate are: H!299 : lung cancer cell and DU145 cells: prostate cancer cells.
If you add protease inhibitor when making the lysates, aliquot them and store them at -80°C, you can use the aliquots for years.
Hi,
I have used degradation-prone proteins for a couple of months with the activity being still intact. This was with purified proteins. Cell lysates might be less stable due to protease action, I assume.
What is the level of protein structural disorder in the HDACs ? B'cos the stability is dependent on the level of protein disorder and exposed loops etc.......You can easily enter your protein sequence in Disembl, a protein disorder prediction tool, to get the lability of the protein in relation to structural disorder.
Still, in western blots antibodies bind to linear epitopes mostly (can bind to conformational epitopes too after renaturation via membrane transfer), and therefore, as long as the protein is intact (even in a linear form) it should be able to give a signal.
Do read this paper if you have time - http://www.nature.com/icb/journal/v85/n1/full/7100004a.html
Best Wishes
Dilantha
Hello,
I have successfully used tissue lysates (various cancer biopsies) prepared several months prior to WB, for detection of multiple proteins. I keep the lysates frozen at -80 deg C. So, storage conditions are important.
Good luck with your experiment!
Hi
Once you have protease inhibitors in your cell lysis buffer and you have stored your lysate at -80 degrees, I believe your protein of interest will be fine. Just avoid frequent freezing and thawing
If the lysate if stored with towing repeatedly it would contain good amount of protein depends on initially concentration too. still there is some degradation due to proteases.
i suggest to do western blotting following the protein extraction once the procedure is standarized
If you add protease inhibitor when making the lysates, aliquot them and store them at -80°C, you can use the aliquots for years.
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