Western blot control - Suggestions? (Nov/07/2008 )
Hello,
I do a Western blot for a protein that has 250kDa ( in HeLa cells). I used to run 8%SDS-gel. But to get a good resolution of my protein I could not use GAPDH as a loading control (from the same gel), because this protein was too smal. Can anyone have idea what to use as a control having about 150kDa?
Thank you in advance.
-alchemic-
you can run a gradient gel to resolve both proteins (5-15% may do the trick for you).
-mdfenko-
QUOTE (mdfenko @ Nov 7 2008, 10:29 PM)
you can run a gradient gel to resolve both proteins (5-15% may do the trick for you).
Thank you mdfenko for suggestion, I will try that!!
-alchemic-
QUOTE (alchemic @ Nov 7 2008, 11:39 AM)
Hello,
I do a Western blot for a protein that has 250kDa ( in HeLa cells). I used to run 8%SDS-gel. But to get a good resolution of my protein I could not use GAPDH as a loading control (from the same gel), because this protein was too smal. Can anyone have idea what to use as a control having about 150kDa?
Thank you in advance.
I do a Western blot for a protein that has 250kDa ( in HeLa cells). I used to run 8%SDS-gel. But to get a good resolution of my protein I could not use GAPDH as a loading control (from the same gel), because this protein was too smal. Can anyone have idea what to use as a control having about 150kDa?
Thank you in advance.
as mdfenko suggests using a gradient gel is the standard; for calibrating a Neville plot, you need references around, higher and lower than 250 kDa; you may try myosin II HC (~180 kDa), for higher than 250 kDa you may use kettin, twitchin, projectin or titin...
-The Bearer-