Best nuclease for removing DNA from bacterial lysates?
Dear all, I have had problems using benzonase nuclease to remove DNA from bacterial (E. coli) lysates, from which I am purifying a ssDNA-binding protein. The problem is, that I want to purify a DNA-free protein, and benzonase did work for that, but then of course I need to remove the benzonase from the preparation, because I later want to use this protein in experiments containing DNA, and benzonase proved to be difficult to completely remove. I still had nuclease activity in my purified protein. I am planning to try the cyanase nuclease from Ribosolutions Inc., since they claim it is faster than benzonase or DNase, and is more easily removed after use, with the inactivation resin that they provide with the enzyme. Does anyone have an opinion on what is your favorite nuclease for removing DNA from bacterial lysates, and that can then be removed itself also? DNase, benzonase, or cyanase..? Thank you in advance, Paul Phelan Tufts University School of Medicine Biochemistry Boston ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
Haunted Biologic DuoFlow system?
I think this will be an unusual question. I use a Biologic Duoflow chromatography system (from Bio-Rad) for a lot of protein purification, and lately I have had a bizarre problem with the system. The pumps have been starting by themselves, even in front of my eyes as I watch, and every time they start running at 10 ml/min., all by themselves. This has happened whether the software that controls the system is open or closed, so I have had to power off the pump station whenever I am not running it, to keep it from running by itself without warning. It behaves just fine when I am running a protocol on it, so at least I can still use it, but a few times when I left it powered on, I have come in to find a whole liter of water or buffer emptied into the waste, and the lines are completely empty. Yes I have contacted Bio-Rad about it, and the rep. said over the phone that she never heard of anything like that, but I haven't had anyone come out to look at it, because that costs! a small fortune. The rep suggested that it could be due to condensation inside the pump station shorting out some connections, so I took everything out of the refrigerated cabinet where we keep it, dried it out at room temp. and it ran just fine (no auto-starts), but a while after I put it back into the cold, it started to run by itself again. I don't expect anyone to have a magical answer, but I wonder if anyone else has ever seen anything like this? Paul Phelan Tufts University School of Medicine Boston ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
Expression vector for GST-tagged TEV protease
Hello everyone, I am looking for an expression vector for GST-tagged TEV protease, and after searching through everything on Addgene and ATCC, I am coming up blank. All I can find are vectors for MBP and His-tagged TEV protease. Google doesn't help much either, it keeps leading me to references to vectors with TEV protease cut sites, which is not what I want. Does anyone know of a GST-TEV protease expression vector? Many thanks, Paul Phelan Tufts University Department of Biochemistry ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
Glutathione Sepharose chromatography at below-neutral pH
I have a quick question for everyone: When using glutathione Sepharose (4B Fast Flow from GE) to purify a GST-tagged protein, has anyone used it at pH 6.5 and have it work? I ask because I am trying to purify a DNA-binding protein that is greatly aggregated after the initial glutathione Sepharose column, and I think it might be pH-related. However, I do think the elution of the fusion protein from glutathione Sepharose will still have to be at pH 8.0 for it to work. Thank you, Paul Phelan Tufts University Boston ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
RE: the absorbance value 1.05 at 260/280 of protein solution
You can use the A260/A280 as an indicator of protein purity. Ideally, pure protein should have an A260/A280 value of 0.6, so 1.05 is very high, and indicates that you have nucleic acid contamination (the A260 component is too high). From: methods-boun...@oat.bio.indiana.edu [methods-boun...@oat.bio.indiana.edu] on behalf of Sudheer Sangeetham [sudheer.pb...@gmail.com] Sent: Wednesday, October 28, 2015 7:13 AM To: meth...@oat.bio.indiana.edu Subject: the absorbance value 1.05 at 260/280 of protein solution Hello people What does it mean by the absorbance value 1.05 at 260/280 while measuring the protein concentration at 280 nm ? Thank you in advance -- Sudheer Babu.S Research Fellow Institute of Biochemistry Biological Research Center Szeged,Hungary. ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
Poly-deoxyribose phosphate?
Here is a quick question: Has anyone ever heard of poly-deoxyribose phosphate (i.e. a bare DNA sugar-phosphate backbone without any bases) being commercially available? I have not been able to find any source for it, and I have made some enquiries, but wondered if anyone else has ever hard of it being available. Thanks, Paul Phelan Tufts University School of Medicine Boston ___ Methods mailing list Methods@net.bio.net http://www.bio.net/biomail/listinfo/methods
