Hi, everyone, I am working on transcriptional factor, I want to define what protein bind to my specific DNA, after I got the sample that bound to DNA, and use SDS buffer to boil it, I can find the white pellet in my sample, I have tried to use DNAase, find the pellet was DNA, my question is that if I directly add DNAase in the protein sample, will it affect following WB and when should I add it, before boiling or after?
Thanks in advance.
Since the SDS-PAGE sample buffer is denaturing, the DNase should be added to the sample before it is mixed with SDS-PAGE sample buffer. You also have to add Mg2+.
So it will not affect the WB, but how can I decide how much DNAase should I add? Today I tried that after boiling, the pellet can go away (become clear) if I add DNAase (10ul, 5.45U/ul). Thanks Adam.
As long as DNA is not covalently linked to proteins, boiling protein-DNA mixture with SDS-PAGE sample buffer (Laemmli buffer) is sufficient to release the proteins from DNA. Further, separation of protein from DNA on the SDS-PAGE and detection of proteins by Western blotting should not present any problem.
I agree with Divaker. If the viscosity presents a problem with loading, you can load the samples while they are still very warm after boiling and it should reduce the viscosity.
I think you will have to find out hpw much DNase you need for your application experimentally. Start by adding a relatively small amount, and see if that is sufficient before adding more. You don't want to add a huge amount because you will then see it as a band on the gel and a very heavy band could distort other bands you are trying to see if they are of similar mass.
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