Re: [ccp4bb] Protein-DNA complex crystallization
Curious, how did you assess that your crystals only have DNA? F On Nov 21, 2011, at 8:59 AM, umar farook wrote: Dear All, I have been trying to crystallize protein DNA complex, but all the time i end up with DNA crystals. Even i changed the length of DNA many times but still no complex, DNA only crystallizes! Does anybody has idea, why do DNA crystallize by itself ? My protein behaves very nicely, Dynamic Light Scattering always shows nice values implies homogenous but once i tried to ran acidic native page but it shows little bit aggregated. The protein is highly hydrophilic and soluble, and has only three cysteines, is it there any possibility of aggregation due to cysteine, when overexpressed in E.coli ? and one more thing i mixed protein and DNA together and ran agarose gel to see any gel shift, indeed there is a binding, but when i take the same thing to set drops, only DNA crystals. Kindly suggest me, what could be done. Thanks Regards, Umar Farook.S - Francis E. Reyes M.Sc. 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Protein-DNA complex crystallization
Sent from my Verizon Wireless Phone - Reply message - From: umar farook umarfaroo...@gmail.com Date: Mon, Nov 21, 2011 10:59 am Subject: [ccp4bb] Protein-DNA complex crystallization To: CCP4BB@JISCMAIL.AC.UK Dear All, I have been trying to crystallize protein DNA complex, but all the time i end up with DNA crystals. Even i changed the length of DNA many times but still no complex, DNA only crystallizes! Does anybody has idea, why do DNA crystallize by itself ? My protein behaves very nicely, Dynamic Light Scattering always shows nice values implies homogenous but once i tried to ran acidic native page but it shows little bit aggregated. The protein is highly hydrophilic and soluble, and has only three cysteines, is it there any possibility of aggregation due to cysteine, when overexpressed in E.* coli* ? and one more thing i mixed protein and DNA together and ran agarose gel to see any gel shift, indeed there is a binding, but when i take the same thing to set drops, only DNA crystals. Kindly suggest me, what could be done. Thanks Regards, Umar Farook.S
Re: [ccp4bb] Protein-DNA complex crystallization
What is Kd? Also, in reply to earlier posts: it is sadly common in crystallizing large protein-DNA complexes to go through a couple dozen different duplexes and several dismally-diffracting crystal forms before finding a good one. Phoebe From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of umar farook umarfaroo...@gmail.com) Subject: [ccp4bb] Protein-DNA complex crystallization To: CCP4BB@JISCMAIL.AC.UK Dear All, I have been trying to crystallize protein DNA complex, but all the time i end up with DNA crystals. Even i changed the length of DNA many times but still no complex, DNA only crystallizes! Does anybody has idea, why do DNA crystallize by itself ? My protein behaves very nicely, Dynamic Light Scattering always shows nice values implies homogenous but once i tried to ran acidic native page but it shows little bit aggregated. The protein is highly hydrophilic and soluble, and has only three cysteines, is it there any possibility of aggregation due to cysteine, when overexpressed in E.coli ? and one more thing i mixed protein and DNA together and ran agarose gel to see any gel shift, indeed there is a binding, but when i take the same thing to set drops, only DNA crystals. Kindly suggest me, what could be done. Thanks Regards, Umar Farook.S
