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Protein of 460 kD on western blot

Electrophoresis

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3 replies to this topic

#1 TanjaMina

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Posted 22 April 2013 - 01:55 AM

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

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Posted 22 April 2013 - 04:23 AM

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.
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#3 TanjaMina

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Posted 22 April 2013 - 06:02 AM

Thanks! I will try! Posted Image

#4 mdfenko

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Posted 23 April 2013 - 03:52 AM

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).
talent does what it can
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