Hello,
I am getting nonspecific bands in HIF-1alpha blot. Can any one suggest why and how I can improve this?
I am using the following conditions
Cell: HCT116
Gel: 8%
Blocking: 5% non fat milk powder
Primary antibody mouse: 1:1000 in plan TBST Santa Cruz
Secondary Antibody: 1:4000 in plan TBST
Hi Manish,
In my experience HIF1 alpha was always a bit of trouble to blot for. Right now we use the BD bioscience antibody #610959 (1:1000 over night at 4oC); however i find it works more consistently when I prepare my samples under hypoxia.
I have heard that HIF become degraded rapidly once the cells are exposed to 21% oxygen. To avoid this, PBS wash buffer is stored in the hypoxia chamber with the cells perminantly. Then the following lysis buffer is prepared:
• 6 ml 0.5 M Tris pH 6.8
• 12.5 ml 100% Glycerol
• 10 ml 10% SDS
• 2.5 ml beta-mercaptoethanol
• 0.5 ml 2-1Bromophenol blue
• 18.5 ml water
Add 500uL of this lysis buffer to (PBS) washed cells and scrape off cells.
The only issue with this, is you can not BCA your protein so you must seed identical numbers of cells and load the same volume of lysate. Judging by my tubulin loading controls it is never too far off!
I hope this helps a little!
All the best
*note, we use a hypoxia chamber with gloves attached to allow us to lyse cells under hypoxia.
Our own experience is the antibody. We have tried 3 different vendors and after one year of struggling only one showed the clean bands. Our cells were from sheep and two vendors gave us a big band at 80-90kd which made no sense to us. I will second Okan's comment on this one.
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