Hi,

I use Hif 1 Alpha from NovusBiologicals (100-479) on heart tissue-samples of P1 mice. I also use Ripa buffer to lysate the tissue, containing proteases-inibitor-cocktail-tablets.

I also use 12%-gels and make a semi dry blot.

load is 30 to 40 µg of protein-lysate per well.

Blocking: 5% Milk-powder in TBS-T

I dont have a Problem to detect Hif 1 Alpha, so I estimate your antibody is maybe a kind of degraded in a way or maybe just not working very well, given that the blot looks normal to you, like the marker was transfered like it should be etc. 

Hi, Try to to detect really known protein like Actin or Tubulin to make sure your SDS-PAGE is good. Then try to load more protein 60-80ug "if you have that much"  and plot it with anti-HIF1a with low dilution like 1-200. I hope that would be helpful. Good Luck.

Ahmed 

Hi,

I use anti-HIF-1 alpha mouse, clone H1alpha67 mouse, NB100-105SS NOVUS Biologicals in 1:1000. It works fine on human cells.

I lyse the cells directly in 1x laemmli buffer under reducing conditions. 

Hi

I am also working on Hypoxia (HIF-1 alpha) for past 8 months. Before giving any advice i would like to know few things from you.

1. what you did to ensure that your hypoxia treatment i

Hi, thank you very much fory your kind suggestions. I can easily detect other proteins regulated by HIF1a that means that the hypoxia condition as well as the blotting is working well. Now, i am considering two options;

Hif1a might be degraded before i lyse the cells and add proteinase inhibitors (PI) becuase it takes me around 10-15 mints to spin down the cells. whats your experience, how soon do you add the lysis buffer+PI?

Another posssibliity is that HIF1a antibodies are degraded or not working well. so anyone have experienced using antiHIF1a (AF1935-SP)?

thanks  

 hi

ok , if you checked that hypoxia is generated it's good. As we all know that it's half life is less then 5 mins, so be ensure the fast processing of samples. After treatment transfer your sample immediately over ice to avoid hif-1 degradation. Above all HIF-1 alpha is very difficult to stabilize and it's signal is also very weak, so you must have to use ECL technique to visualize your bands.

thanks-  let me ask - do you guys use nitrocellulose membran or pvdf membran for transfer? is HIF1a is hydrophobic ?

we used PVDF Membrane for transfer.

you earlier said that you checked other proteins regulated by HIF-1 alpha, can you name few of them.

Hi, Dayanidhi, I would like to know few more details regarding your western blot and transfer methods. From mine side, I am giving you the details what we do regularly in our lab for western blot.

Buffer : Phosphate buffer (Not PBS)

Transfer Current = 400mA ( Plz dont follow Volt..strict on current)

Time= 2.5-3hr (at 4 degree C)

Note: Pre-stained markers conforms the transfer efficiency so use it

Hi,

I sometimes see your problem. In my view, some factor can affect on result

your protein is not enough for detecting

time is not enough. I usually wet transfer at 60 mA over night (15hrs) at 4 degree C using tris-glycine transfer buffer. 

I don't know what type of membrane you use. If you use PVDF membrane it is crucial that you have to activate it by methanol before using

i read a document of Abcam. Their RIPA lysis buffer do not include PI. Iam not sure whether PI affect  on your protein or not

Best regard,

Hi Naresh,

we are generally prefer over night transfer of protein to the PVDF membrane at 400 mA in cold room (4 degrees). After western blot we use ECL method for detection. if you have any further queries you can ask to me anytime.

Thank you very much guys..