Western blot with Adipose cells - (Oct/30/2007 )

Hi everyone,

I need to find a lysate buffer for Adipose cells and perform western blot.

I had tried with SDS buffer containing:
- 1% SDS
- 10mM EDTA (pH 8.0)
- 50mM Tris-HCl (pH 8.0)
but I was not sure with this condition - could or not - inhibit some proteases, especially phosphatase.

When I lysed adipocytes with SDS buffer, the sample always became sticky. Thus, I needed to boil it at 99oC for 10 min.

I also tried with RIPA buffer containing inhibitor cocktail for protease, but I got difficulty to remove the lipid layer.

I hope anyone who has experience with Adipocytes can help me.

Hi everyone,

I need to find a lysate buffer for Adipose cells and perform western blot.

I had tried with SDS buffer containing:
- 1% SDS
- 10mM EDTA (pH 8.0)
- 50mM Tris-HCl (pH 8.0)
but I was not sure with this condition - could or not - inhibit some proteases, especially phosphatase.

When I lysed adipocytes with SDS buffer, the sample always became sticky. Thus, I needed to boil it at 99oC for 10 min.

I also tried with RIPA buffer containing inhibitor cocktail for protease, but I got difficulty to remove the lipid layer.

I hope anyone who has experience with Adipocytes can help me.