Re: [ccp4bb]: Protein_DNA complex

[matthew hogg <Matthew.Hogg@uvm.edu>]

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>>

I have had good luck in picking out some crystals, washing them, 
dissolving them in PNK buffer, end labeling them with P-32, and then 
separating DNA bands via 18% SDS-PAGE This has proven useful in seeing 
single nucleotide differences between the starting DNA and the DNA from 
the crystal.

I have also done the same thing with fluorophore labeled DNA 
(fluorescene) by scanning the gel with our Bio-Rad Molecular Imaging 
system and thus skipped the P-32 step. I just wash the crystals, 
dissolve them, load them on the gel and run it.

Matthew Hogg
Lowly Graduate Student
Microbiology and Molecular Genetics
University of Vermont
Burlington, VT  05405
802 656-9533

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>>
>> Folks,
>>
>> Is there a clever way to tell if our crystals are complexed 
>> protein:DNA
>> versus unbound protein, without having to wait until we have maps? We 
>> have a
>> fluorophore attached to the DNA fragment.
>>
>>
>> Elias J. Fernandez, PhD
>> Assistant Professor
>> University of Tennessee
>> Biochemistry, Cellular and Molecular Biology
>> Knoxville, TN 37996-0840
>> Tel. No. 865-974-4090
>> Fax. No. 865-974-6306
>> e-mail: elias.fernandez@utk.edu
>>