Gradient - (Mar/16/2015 )

Hi,

I need to see on the same gel one protein around 80 kDa, another one around 60 kDa, p53 and a 15 kDa histone.

My boss told me to run a gradient Western Blotting or something like that. But I have done only classical Western Blotting and I have no idea how to do this.

Can anyone please explain me the protocol?

Thanks.

Gradient gels are just like regular gels, but have a high % gel at the bottom and a low % at the top, thus allowing you to separate widely different sizes. There are specialist equipment for pouring gradient gels (you can also buy them pre-made, if you have the equipment to hold the commercial gels, Life technologies and Biorad are two commonly used ones), so check in your lab and others around to see if you have the right equipment. Once you have poured the gel, the rest is the same as regular WB.

A basic equivalent is to pour a thin strip of 15% in the bottom of a casting set, then a strip of 10%, followed by a 7% then 4%, or some similar percentages.