Bluenoise Posted November 22, 2005 Share Posted November 22, 2005 Okay so I'm studying this lil calcium binding protein. And being a crazy canadian I've decided to make a who bunch of highly mutated versions and see what effect it has on this proteins function. But it seems when you make these mutants this protein gets really friggin hard to purify from DNA. I suspect it's wrapping around the DNA helices. Now the problem is I really need to get rid of this DNA so that when I do my assays there's no question of interferance from the DNA, (it's not likely to happen but I can't take the chance) so I can write my paper. So if anyone know this really good trick for getting rid of DNA I'd really appreciate it. Oh btw this is a pretty hardy little protein it can easily be boiled to 80 C without any degradation. I've already run done an ammonium persulfate cut and ion exchance on an FPLC. But I can't seem to get rid of this freaken DNA. I do have quite a large volume of protein so I have some room to experiment with different methods. I'm considering doing some size exclusion chromatorgraphy. But I think it would be best to try some chemical methods first. Link to comment Share on other sites More sharing options...
RyanJ Posted November 23, 2005 Share Posted November 23, 2005 I suppose a centrifuge could work to seperate out the strands with different masses, not shure how good or even if it would work correctly though. Cheers, Ryan Jones Link to comment Share on other sites More sharing options...
Bluenoise Posted November 23, 2005 Author Share Posted November 23, 2005 Well yeah I'd lover to be able to do that but... ...to centrifuge out the DNA I need a way to first cause it to percipitate out of the solution, you can't centrifuge out DNA in solution. Alcohol wont work since that would contaminate my protein with alcohol. So if anyone knows some knifty dna percipitation compound that I could then remove easily. Like maybe a salf that I could dialyse out. Link to comment Share on other sites More sharing options...
ecoli Posted November 23, 2005 Share Posted November 23, 2005 Couldn't you use DNase? That's what I used when isolating RNA... perhaps it could work for you too. Link to comment Share on other sites More sharing options...
Bluenoise Posted November 23, 2005 Author Share Posted November 23, 2005 It's been tried without much sucess. Also we'd still need to get the nucleotides out after that. If we're right about our protein wrapping around DNA than that would sheild it from DNase anyways. Plus our sample of protein is pretty large and concentrated. The idea of contaminating it with another protein is very unappealing. *edit* Hmmm I'm thinking I'll might be able to try percipitating it with protamine sulphate. Anyone know if this can cause problems with proteins? *edit #2* Looks like protamine sulphate really only percipitates basic or large protein complexes. Seeing as how mine is ultra small and acidic this may work... Link to comment Share on other sites More sharing options...
ecoli Posted November 23, 2005 Share Posted November 23, 2005 what about Ammonium sulfate or Sodium sulfate? Link to comment Share on other sites More sharing options...
Bluenoise Posted November 23, 2005 Author Share Posted November 23, 2005 If you read my initial post you'll see I've already done that in my protein purification step. Still have DNA. Link to comment Share on other sites More sharing options...
ecoli Posted November 23, 2005 Share Posted November 23, 2005 If you read my initial post you'll see I've already done that in my protein purification step. Still have DNA. oops, sorry about that. Link to comment Share on other sites More sharing options...
Bluenoise Posted November 23, 2005 Author Share Posted November 23, 2005 oops, sorry about that. Oh no worries man, I appreciate any suggestion I can get! The input is definatley needed. Link to comment Share on other sites More sharing options...
Yggdrasil Posted November 23, 2005 Share Posted November 23, 2005 It may be worth trying to purify the protein under denaturing conditions (i.e. 6M urea) and refolding it (by dialyzing out the urea) once it has been purified from the DNA. When denatured, your protein shouldn't bind DNA so it should be fairly simple to remove the DNA (especially if you have an affinity tag on your protein). Since your protein is pretty small, you should recover good yields of folded protein when you dialyze out the urea. Link to comment Share on other sites More sharing options...
Bluenoise Posted November 23, 2005 Author Share Posted November 23, 2005 Yeah I was considering that, but I'll save that as a last resort. There's no affinity tag, the nature of our proteins funtion doesn't really allow for one. (It's small, linear and interacts along its whole length) I'm just a bit woried with all the modifications I've made to the protein that it wont refold and agrigate instead. but if all else fails I'll definatley give that a shot. Thanks. Link to comment Share on other sites More sharing options...
Skye Posted November 23, 2005 Share Posted November 23, 2005 Maybe heat to ~70, melt DNA, add some precipitating agent, then let it cool. Link to comment Share on other sites More sharing options...
Bluenoise Posted November 23, 2005 Author Share Posted November 23, 2005 Hey thanks Skye that's interesting that you said that. I acutally proposed using heat to melt the DNA and then seperating it to a PHD student in my lab and he looked at me like I was crazy. Though my idea was to pass it hot through a column. It is possible but tricky. Percipitation might be easier, but it may also be tricky getting the right concentration of salts as solubility will change with temp. But we both can't be crazy right? lol I'll definaltey try that. It looks like I have a few things I can try. I'll give them a shot tomorrow probably (assuming I have the supplies in lab now) and tell you guys about the results. Link to comment Share on other sites More sharing options...
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