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SDS-DNA-PAGE - How to? (Jun/23/2009 )

Hi, I'm new to the BioForum (as a user), even though I've been here before looking for answers, English isn't my native languaje so...you know.

I've been working with an endonuclease (recombinant), and we have the enzyme "purified" by Ni-NTA, I say "purified" because we have some problems and the enzyme is not totally pure, there's still some rubish not some much, but is there anyway, even though my enzyme is pretty abundant and the band is totally clear . When I test the enzyme, with tango buffer for example, and with a plasmid, the enzyme is capable of degrade a ug of DNA in two hours at 37 C and at RT. We're trying to do a SDS-DNA-PAGE, to confirm further any doubt that the band of the size of our Endonuclease is the responsible of the DNA degradation and not the other faint bands that we observed in the SDS-PAGE with coomassie. Basically the SDS-DNA-PAGE is an normal SDS-PAGE gel with DNA on it (on the running). In the protocol says that the DNA to use is the Salmon Sperm DNA added to the solution of acril/bis, SDS, H2O, PSA and TEMED at a final concentration of 1mg/ml, I tried to dissolve the salmon DNA in water without any success, so since it was the first experiment I decided to use a plamid that i had at a high concentration. After the gel is run, it is submerged in a buffer (TEM; Tris, EDTA, MgCl2) overnight, to dilute the SDS allowing the protein to renature and test its endonuclease activity, then the gel is stained by EtBr for 30 minutes and if the protein is active, you would see an empty space where the band is supposed to be an the rest of the gel glowing like hell. Of course, you have to run another gel at the same time and under the same contidions but stained with coomassie to compare. As you may infer by now, the experiment was not successful, and some questions arise.


<*>If you leave the gel in that buffer all night long, without the fixing solutions that are present in the common coomassie stain...are my proteins still there or they difusse away?
<*>Does any body know how to dissolve the Sperm Salmon DNA
<*>Can anybody help me?
<*>What else can I do to confirm that is my protein the one that is degrading the DNA?


Thanks in advance, and greetings from Colombia!

-hineiko-

hineiko on Jun 23 2009, 12:09 PM said:


<*>If you leave the gel in that buffer all night long, without the fixing solutions that are present in the common coomassie stain...are my proteins still there or they difusse away?
<*>Does any body know how to dissolve the Sperm Salmon DNA
<*>Can anybody help me?
<*>What else can I do to confirm that is my protein the one that is degrading the DNA?

1. the protein may diffuse some and give a large, blurry band and some will leave the gel. but enough should remain in the gel. you may want to add some (0.1%) triton x-100 to the renaturing buffer. it displaces sds from the protein and facilitates renaturing.

2. dry dna is notorious for difficulty in solubilizing. you can warm the solution while stirring to solubilize. it may still take a while so do this in advance of preparing the gel.

4a. you can try running a native gel (without sds) in a similar manner. then you won't have to renature.

4b. you can run the protein on gel filtration and show that the activity is in the peak fractions with your protein.

3. i hope i did.

-mdfenko-

Thanks, for the tips, I will definitely try the Triton. But now I'm focused on the salmon sperm dissolution, I'm getting frustrated!

-hineiko-

hineiko on Jul 2 2009, 04:02 AM said:

Thanks, for the tips, I will definitely try the Triton. But now I'm focused on the salmon sperm dissolution, I'm getting frustrated!


Hi i used to dissolve SS-DNA in aqua dest to a concentration of 10 mg/ml, adjust pH to 7. Stir for several hours and then sonicate (30 cycles, 30secs sonication, 20secs rest). Recalculate the concentration
Good luck

-fysio lab-