3 replies to this topic

[TanjaMina]

How to detect DNA PK-cs, protein of 460 kD by western blot?! Which percentage of gel to use?!

[mdfenko]

i would use either a 5% gel or 5-15% or 4-12% gradient gel (depending on whether you want to see smaller proteins, as well).

add 0.05% sds and 10-20% methanol to the transfer buffer to facilitate transfer of high molecular weight proteins.

you can download a very useful handbook on western blotting (as well as other handbooks) from ge healthcare life sciences.

[TanjaMina]

Thanks! I will try!

[mdfenko]

one more thing,

is the protein a single subunit or multiple subunits? if multiple then the apparent mw of the subunits will be less than 460 kDa on sds-page. you may want to adjust the gel percentage based on the subunit size(s) rather than the native size (unless you are running a native gel).