Help! questions on DNA pull-down assay - (May/28/2008 )

Hello, there
This is my first time to post here. I am very excited to see there are so many warm-hearted scientists to provide good suggestions.
I have been trying to use a 600bp DNA probe to pull down proteins from cellular extract. The system is the simple affinity purification via biotin-streptavidin interaction. Our goal is to analyze the eluted proteins by mass spec.

First, I immobilize the biotin-modified DNA probe onto streptavidin-coated Dynabeads. Then, apply cellular extract onto the complex. After wash, boil the beads with 2% SDS. Now, here is the problem. I got a lot of smearing on the silver-staining gel. It was from the probe. Normally, the probe would not be able to see because it would be very small. But I have a DNA fragment with 600bp. Ok, I then used DNase I to cut the DNA probe, thinking that with this step, I would get specific elution. The silver staining gel suggested that a lot of proteins were released upon the DNase I treatment (Fraction1). Meanwhile, there were a lot of proteins stuck on the beads (Fraction2) as well. I thought Fraction 1 should contain most DNA binding proteins. But after I checked with Western Blot, to my surprise, all the DNA binding proteins (known to bind to the DNA sequence specifically) that have been chosen to verify the system somehow still remained on the beads. Follow-up Agarose gel analysis confirmed that most DNA probe was cleaved. Why on the earth would the DNA binding protein get stuck on the beads then? Could someone kindly provide me with some explanations and comments, please? Many thanks!!!
P.S.The negative control, in which no DNA probe was added, showed absolutely no proteins being pulled down.

My guess is that the DNA-binding proteins protected the DNA from DNA cleavage by DNase. Like footprinting.

Did you get the smear in fraction 2?
Could the smear be streptavidin?
Can you elute the proteins with something other than SDS? NaCl, KSCN, Urea, Biotin?

dan

Dear Dan,
Thank you very much for your comments. But I do not think I have the problem figured out. The reason is if the DNA band is gone in Fraction2, it means that DNase I is able to cleave the DNA probe. Therefore, it is logical to assume that most DNA binding proteins should be eluted in Fraction 1. However, western blot results showed that most of those proteins remain on the beads. This is confusing. Because if the proteins are fished out by the probe, when the probe is destroyed, why would the protein still get stuck on the beads? (the DNA probe is 5'-biotinylated, the beads are streptavidin coated).

Thanks

What I was thinking is that all of the unbound DNA is cleaved by DNaseI, so maybe you get rid of 95% of the DNA. The remaining bead-bound DNA is protected from cleavage because it is tightly bound by proteins.

I guess, if you are worried that the fraction2 proteins are not DNA-binders, compare them to fraction 2 proteins from streptavidin beads alone (neg control). And if you worry that some interesting proteins elute in fraction 1, isolate them too.

Sounds like the assay is working, based on your western results. you might also probe for DNA-binders that are not sequence-selective, such as RPA, to get an idea how selective your assay really is.

good luck
dan

What I was thinking is that all of the unbound DNA is cleaved by DNaseI, so maybe you get rid of 95% of the DNA. The remaining bead-bound DNA is protected from cleavage because it is tightly bound by proteins.

I guess, if you are worried that the fraction2 proteins are not DNA-binders, compare them to fraction 2 proteins from streptavidin beads alone (neg control). And if you worry that some interesting proteins elute in fraction 1, isolate them too.

Sounds like the assay is working, based on your western results. you might also probe for DNA-binders that are not sequence-selective, such as RPA, to get an idea how selective your assay really is.

good luck
dan

You are using a too high concentratin of SDS for elution! Boiling in 2% SDS will break the streptavidin-biotin bond. Try using 0.1% SDS instead. That will elute your protein and leave the NA on the beads.