I am doing how the hypoxia affect tight junction in retinal pigment epithelial cell (RPE), especially, breaking the RPE layer by hypoxia.
I chose to use CoCl2 for inducing hypoxia and performed the cell viability test depending on dose of CoCl2 and treatment time.
And 500uM for 72h could be used to reduce cell viability which means it could break the RPE layer.
However, I can not see any band of HIF-1a in western blotting, both in control group and 500uM 72h group.
I searched the paper about RPE and CoCl2, and what I see is that most of researchers saw the HIF-1a band within 24h.....
Is the HIF-1a is disappeared over 24h?
Now this is the problem, 24h is slightly weak for breaking RPE layer (the cell viability is not so low compared to untreated cell).
Should I treat more higher concentration of CoCl2 for lowering cell viability within 24h?
Personally I've not really used CoCl2 to induce HIF1, but I know for sure that collection of cell lysates is a major bottle neck of detecting HIF1a protein using western blot, as HIF1a degrades very quickly.
The way I did it was using hypoxic chamber and actually collecting cell lysates within the chamber and on ice. I would presume you are removing the CoCl2 containing media, rinse with PBS and then lyse the cells? In that case you can try keeping the cells on ice and rinsing with ice-cold PBS, and make sure your lysis buffer has protease inhibitors. Try also to keep it within 30 seconds as that seems to be the time it takes for the protein to degrade (in my experience).
Hi Mr Kim. Have you checked the total protein? Is your transfer protocol optimised for HIF1 detection? How about actin - are the bands present? If the problem lies in the nonexistence of bands, we must consider whether the proteins are successfully transferred onto the membrane or not.
Dear Chang, thanks for your kind answer.
I didn't know about degradation of HIF-1a.
I washed my cell with warm PBS and lysed cell with RIPA with protease inhibitor cocktail and wait for 5 min on the ice.
I will do with ice cold PBS.
But one more question. the 30 sec means, the lysis protocol done in 30 sec?
RIPA buffer require the time at least 3min. Should I use the cell scraper?
Dear Rahman, yes I saw the beta-actin band but sadly nothing in HIF-1a.
I also checked the gel after transfer, if there was remaining band which is untransfered, using coomassie blue staining. But nothing left in gel, so the whole band is transfered to my PVDF membrane I think...........
30 seconds refer to between you taking cells out of the media/chamber and the moment you add RIPA buffer. I would always use a cell scraper in this case! Once you have the samples on ice and in lysis buffer with protease inhibitors it's not as urgent :)
That's sound good. I think I have to lysis the cell within 30sec..
my rule of thumb when it comes to HIF proteins is: put the cells on ice and do all the PBS washing/RIPA scraping on ice. Good luck!
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