Concentration of EDTA in lysis buffer - (Dec/08/2008 )
I'm doing western for hemeoxygenase-1 (HO-1). My lysis buffer is mainly protease inhibitors cocktail from Sigma, which contains 1 mM EDTA already, so I didn't add additional EDTA. Googling results: people use either 1, 2, or 5 mM EDTA for HO-1 western.
Qs: Is there any special considerations for HO-1 in terms of components of lysis buffer? what concentrations of EDTA are you guys using no matter what protein of interest you are dealing with? Has anybody ever seen degraded proteins on the gel due to insufficient amount of lack of EDTA (striped/streaked bands)?
Happy Christmas!
Hanhan
-hanhan2008-
QUOTE (hanhan2008 @ Dec 8 2008, 11:50 AM)
I'm doing western for hemeoxygenase-1 (HO-1). My lysis buffer is mainly protease inhibitors cocktail from Sigma, which contains 1 mM EDTA already, so I didn't add additional EDTA. Googling results: people use either 1, 2, or 5 mM EDTA for HO-1 western.
Qs: Is there any special considerations for HO-1 in terms of components of lysis buffer? what concentrations of EDTA are you guys using no matter what protein of interest you are dealing with? Has anybody ever seen degraded proteins on the gel due to insufficient amount of lack of EDTA (striped/streaked bands)?
Happy Christmas!
Hanhan
Qs: Is there any special considerations for HO-1 in terms of components of lysis buffer? what concentrations of EDTA are you guys using no matter what protein of interest you are dealing with? Has anybody ever seen degraded proteins on the gel due to insufficient amount of lack of EDTA (striped/streaked bands)?
Happy Christmas!
Hanhan
For Xenopus oocyte/embyro lysis I use 1mM EDTA, but for cell culture lysis I do not add any. In both cases I use 1x Complete Mini Protease Inhibitor Cocktail (Roche).
-nelsens-