Hello All,
I am working with HepG2 cell lines and will be using AMPK alpha AB from rabbit and GAPDH AB from rabbit as my primary antibody for western blot. What marker should i use on my SDS-PAGE gel before using it for the western blot and what concentration of acrylamide and TEMED should i use as well?
Thanks
AMPK alpha is around 63-64 kDa.
and GAPDH is arounf 36 kDa
i.e during western blotting you must be able to see distinct bands at this molecular weight.
you may use a prestained ladder. we use genedirex blueye ladder in our lab.
to separate both of your proteins, it is advisable to use a 12% SDS page gel.
The bright red band would be visible at around 75 kDa and the green band would indicate 25 kDa which brightly fluoresce on secondary antibody incubation and can be used as markers for you desired protein.
depending on the volume of the acrylamide required, you may use TEMED accordingly.
For 15 ml resolving mixture the composition is:
Distilled water: 4.9 ml
30% acrylamide : 6 ml
1.5M Tris (pH 8.8): 3.8 ml
10% SDS: 150 ul
10% APS: 150 ul
TEMED: 10 ul
Hope it helps.
Good luck!
AMPK alpha is around 63-64 kDa.
and GAPDH is arounf 36 kDa
i.e during western blotting you must be able to see distinct bands at this molecular weight.
you may use a prestained ladder. we use genedirex blueye ladder in our lab.
to separate both of your proteins, it is advisable to use a 12% SDS page gel.
The bright red band would be visible at around 75 kDa and the green band would indicate 25 kDa which brightly fluoresce on secondary antibody incubation and can be used as markers for you desired protein.
depending on the volume of the acrylamide required, you may use TEMED accordingly.
For 15 ml resolving mixture the composition is:
Distilled water: 4.9 ml
30% acrylamide : 6 ml
1.5M Tris (pH 8.8): 3.8 ml
10% SDS: 150 ul
10% APS: 150 ul
TEMED: 10 ul
Hope it helps.
Good luck!
Hello, For GAPDH the marker is at about 36-37 kDa.
AMPK alpha is in the around 63 kDa. the gel percent is 8%.
Good luck!
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