I am intending to stabilise HIF 1 aplha expression in cancer cells; I mean to say to induce hypoxia mimetic environment. Since we don't have a hypoxic chamber availability, we are intending to go with 100 micro Molar Cocl2 treatment for 24 hrs...
I have certain concerns about Cocl2 stability;
1) What is the optimum temperature to store the Cocl2 stock for longer duration without any degradation to it?
2) Shall I have to add Cocl2 from my stock to the culture media a fresh each time or I can make a working concentration of Cocl2 (100 Micro M) in culture media and store it at 4 degrees?
Any suggestions will be highly acknowledged....
Thanking you in advance
Dear Rayees
According to sigma, the storage temperature for CoCl2 is 2-8°C (Link). However, It is good to look the CoCl2 properties from company that u would like to purchase it.
For ur second question: please hv a look on attached file. It is CoCl2 protocol in details. In my opinion you can store CoCl2 at 4 degree and for each experiment prepare a fresh 100 mM stock solution in PBS. It is the safest one ;)
http://www.sigmaaldrich.com/catalog/product/sigma/15862?lang=en®ion=IR
Thanks Fatemah
Thanks a lot for your suggestions. However, I have one more doubt, " Can we store the harvested cell pellet at -80 degrees and later on go for lysis of pellet to obtain total protein and subject it to SDS-PAGE and Western blot?
Dear Rayess,
I have been inducing hypoxia in many types of human cell lines and we usually prepare fresh Cocl2 every time. The stock as mentioned earlier can be saved at 4 degrees but its not good to save it for longer than 3-4 months.
Regarding your second query in case you are trying to do western for hypoxia associated proteins or any other proteins whose half life is too low I will suggest you to make lysates and add appropriate amount of protease inhibitor in it to prevent protein degradation and then you can save in -80. I have used lysates older then a year and it has worked fine for me.
Good luck!
Thanks Karuna Mittal,
Thanks karuna Mittal for your suggestions. However, I would bother you once more, is there any problem to store the harvested cell pellets at -80 degrees, and say eg., after a month or so make the protein lysate out of the stored pallets and proceed for western blot of Hypoxial marker proteins,???
Any suggestions would obviously help me....
Thank you
Hello Rayees,
In general there is no problem in flash freezing the cell pallets but if you are looking at the fractional proteins (like nuclear and cytoplasmic which we generally do when we look for HIF) the flash freezing of pallets can be a problem. The flash freezing can cause the piercing of nuclear membranes by ice crystals formed and that can effect the nuclear protein separation. I personally work with hypoxia a lot and in past three years of my experience I have got best results with fresh lysates. Good luck!
Karuna Mittal
- Badges
- Science topic
- Similar topics
- Medicine
- Oncology
- Cancer Research