[ccp4bb] how to quantitate protein which dont have ne aromatic residue
hello everybody i am working on the protien which dont have any aromatic residue i do fplc other purification using 220 absorption, but i want to quantitate protein precisely i have tried using BCA nd bradford but both methods quantification is not matching,,so any one is having sum idea how to quantitate it precisely thanks in advance for your valuable suggestion.. Arpit Mishra
Re: [ccp4bb] how to quantitate protein which dont have ne aromatic residue
you can do amino acid analysis on your pure protein, using a commercial or academic service - I hope these are still around. You should only need to do this once, then relate the result to your A220, BCA and Bradford assays. Mark Quoting Arpit Mishra: hello everybody i am working on the protien which dont have any aromatic residue i do fplc other purification using 220 absorption, but i want to quantitate protein precisely i have tried using BCA nd bradford but both methods quantification is not matching,,so any one is having sum idea how to quantitate it precisely thanks in advance for your valuable suggestion.. Arpit Mishra Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] how to quantitate protein which dont have ne aromatic residue
It is not surprising that your bradford and BCA assays don't agree if you have no aromatic amino acids in your protein. Bradford dye binds to hydrophobic residues, mainly aromatics, so I would guess your bradford is consistantly giving lower measurements than the BCA assay. I also wouldn't be surprised if the results of your Bradford vary significantly between replicates. The BCA assay reagent interacts with the backbone amides, not with any sidechains, so I would tend to believe that measurement more than anything else you have done. I work with a protein that has very few hydrophobics (only one aromatic - a Phe) and I have found that Bradfords are unreliable, but the BCA assay tends to be consistent. Mike - Original Message - From: Arpit Mishra ar...@igib.in To: CCP4BB@JISCMAIL.AC.UK Sent: Saturday, April 9, 2011 2:52:21 AM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] how to quantitate protein which dont have ne aromatic residue hello everybody i am working on the protien which dont have any aromatic residue i do fplc other purification using 220 absorption, but i want to quantitate protein precisely i have tried using BCA nd bradford but both methods quantification is not matching,,so any one is having sum idea how to quantitate it precisely thanks in advance for your valuable suggestion.. Arpit Mishra -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] how to quantitate protein which dont have ne aromatic residue
Michael Thompson mi...@chem.ucla.edu a écrit : There is a very simple and very quick method that yields an answer approx. 15% reliable: measuring the increment of index of refraction due to the protein. The measurement of an index of refraction can be very accurate. You only need something like a 5µl drop at 1 mg/ml (the order of magnitude should be correct...). Unfortunately, a refractometer is not common in biology labs, but this is a very valuable method. The link between the increment of index of refraction and the protein conc. can be found easily on the web. Philippe Dumas It is not surprising that your bradford and BCA assays don't agree if you have no aromatic amino acids in your protein. Bradford dye binds to hydrophobic residues, mainly aromatics, so I would guess your bradford is consistantly giving lower measurements than the BCA assay. I also wouldn't be surprised if the results of your Bradford vary significantly between replicates. The BCA assay reagent interacts with the backbone amides, not with any sidechains, so I would tend to believe that measurement more than anything else you have done. I work with a protein that has very few hydrophobics (only one aromatic - a Phe) and I have found that Bradfords are unreliable, but the BCA assay tends to be consistent. Mike - Original Message - From: Arpit Mishra ar...@igib.in To: CCP4BB@JISCMAIL.AC.UK Sent: Saturday, April 9, 2011 2:52:21 AM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] how to quantitate protein which dont have ne aromatic residue hello everybody i am working on the protien which dont have any aromatic residue i do fplc other purification using 220 absorption, but i want to quantitate protein precisely i have tried using BCA nd bradford but both methods quantification is not matching,,so any one is having sum idea how to quantitate it precisely thanks in advance for your valuable suggestion.. Arpit Mishra -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] how to quantitate protein which dont have ne aromatic residue
At 9:47 AM -0700 4/9/11, Michael Thompson wrote: Bradford dye binds to hydrophobic residues, mainly aromatics, The statement above is not accurate. Compton and Jones. Anal. Biochem. 151(2): 369-374, 1985 - John --
Re: [ccp4bb] how to quantitate protein which dont have ne aromatic residue
It is not surprising that your bradford and BCA assays don't agree if you have no aromatic amino acids in your protein. Bradford dye binds to hydrophobic residues, mainly aromatics, so I would guess your bradford is consistantly giving lower measurements than the BCA assay. I also wouldn't be surprised if the results of your Bradford vary significantly between replicates. The BCA assay reagent interacts with the backbone amides, not with any sidechains, so I would tend to believe that measurement more than anything else you have done. I work with a protein that has very few hydrophobics (only one aromatic - a Phe) and I have found that Bradfords are unreliable, but the BCA assay tends to be consistent. Bradford reagent is colloidal Coomassie G-250 and it's binding to proteins is very complex, depending on local structure, hydrophobic interaction and basic charges (mainly Arg residues). So yes, it is quite variable protein to protein but it is not a simple function of aromatics. - Dima
[ccp4bb] immobilized DNA resin
Hello ccp4 enthusiasts: I am afraid this is a non-ccp4 related question. Can anyone recommend an immobilized dsDNA chromatographic resin for purification of DNA-binding proteins? GE seems to have something - I was wondering if people have other recommendations? In the age of GST and His tags etc., these are not very much used, but I do not have a tag in this case... Thanks a lot, Alex
Re: [ccp4bb] immobilized DNA resin
Hi Alex, Most DNA-binding proteins has decent affinity to heparin columns. Have you tried purifying your protein over heparin columns? Cheers, Raji On Sat, Apr 9, 2011 at 8:44 PM, Alexandra Deaconescu deac...@brandeis.eduwrote: Hello ccp4 enthusiasts: I am afraid this is a non-ccp4 related question. Can anyone recommend an immobilized dsDNA chromatographic resin for purification of DNA-binding proteins? GE seems to have something - I was wondering if people have other recommendations? In the age of GST and His tags etc., these are not very much used, but I do not have a tag in this case... Thanks a lot, Alex -- --- Raji Edayathumangalam Research Fellow in Neurology, Harvard Medical School Postdoctoral Fellow, Center for Neurologic Diseases, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
Re: [ccp4bb] how to quantitate protein which dont have ne aromatic residue
John, I believe my statement is accurate. The Compton Jones paper you cite states in the abstract: Interactions are chiefly with arginine rather than primary amino groups; the other basic (His, Lys) and aromatic residues (Try, Tyr, and Phe) give slight responses. The binding behavior is attributed to Van der Waals forces and hydrophobic interactions. Also, 23 years later, the following paper provides a detailed study of the mechanism of Coomassie Brilliant Blue G-250 binding to proteins and discusses the hydrophobic interactions I mentioned between aromatic residues and the dye. Georgiou, et al. Anal Bioanal Chem. 2008 May;391(1):391-403. Cheers, Mike - Original Message - From: John A. Newitt newit...@gmail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Saturday, April 9, 2011 12:06:23 PM GMT -08:00 US/Canada Pacific Subject: Re: [ccp4bb] how to quantitate protein which dont have ne aromatic residue At 9:47 AM -0700 4/9/11, Michael Thompson wrote: Bradford dye binds to hydrophobic residues, mainly aromatics, The statement above is not accurate. Compton and Jones. Anal. Biochem. 151(2): 369-374, 1985 - John -- -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
[ccp4bb] immobilized DNA resin
Hi, I have never done this myself, but as far as I know, DNA can be directly conjugated through their primary amino groups to CNBr-activated beads or NHS-activated agarose beads. These beads are supplied by many companies: pierce, sigma, biorad, GE healthcare, etc.. - the same thing used for making protein-conjugated beads through amine coupling. Unmodified DNA works, since the bases contain primary amines. In the early days some people just loaded denatured DNA onto CNBr-activated beads and then they would react. Here is one of the early papers: http://onlinelibrary.wiley.com/doi/10./j.1432-1033.1975.tb04151.x/pdf My idea: if you generate sticky ends with some restriction enzymes or the Klenow fragment, it should help exposing the bases on the overhangs, then you should not need to denature the DNA and worry about all the crazy ways that the DNA sits on the beads. Having said that, ideally, terminally amine-labeled oligos should be used whenever possible, as it is more likely to gives you higher degree of coupling and site-specific conjugation. Only one of the two chains needs the NH2 label, then the two complementary oligos can be annealed to make a one-end labeled dsDNA. When a longer piece is needed, the dsDNA can be generated by PCR with one 5'amine labeled primer and one regular primer. You can synthesize amine labeled oligos from most oligo synthesis facilities. Here is a literature discussing such coupling through amine modified oligos: http://www.plosbiology.org/article/info:doi%2F10.1371%2Fjournal.pbio.0020173#pbio-0020173-g003 Amine coupling is discussed in the part Oligonucleotide Hybridization Chromatography. Zhijie -- From: Alexandra Deaconescu deac...@brandeis.edu Sent: Saturday, April 09, 2011 8:44 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] immobilized DNA resin Hello ccp4 enthusiasts: I am afraid this is a non-ccp4 related question. Can anyone recommend an immobilized dsDNA chromatographic resin for purification of DNA-binding proteins? GE seems to have something - I was wondering if people have other recommendations? In the age of GST and His tags etc., these are not very much used, but I do not have a tag in this case... Thanks a lot, Alex
[ccp4bb] off topic: Is deglycosylation necessary for crystallization?
Hi Engin, Thanks for the correction. This saved a lot of fancying of mine for moving to insect cells :-). Let me also add a little on the yeast side (no personal empirical evidence either): wt yeasts generate hypermannosyl N-glycans, which are tens of to more than a hundred mannose (or modified Man) residues added to the mannose core, forming a huge N-glycan. I do not know if these hypermannosyl N-glycans are still sensitive to endo H, but at least I think some people have already complained a lot about this hyperglycosylation, due to all kinds of concerns. To my memory, there was a company that mutated the mannosyltransferases responsible for the polymerization of mannose in pichia pastoris. Unfortunately, now I cannot find this information again. I wonder if any crystallographer here can share their experience with respect to these mutant pichia strains. Zhijie From: Engin Özkan Sent: Saturday, April 09, 2011 10:44 PM To: Zhijie Li Subject: Re: [ccp4bb] off topic: Is deglycosylation necessary for crystallization? On 4/8/11 1:43 PM, Zhijie Li wrote: 5. Finally, to my knowledge, proteins produced from insect cells are mainly high-mannose type (I never experimentally tested this though, just took this idea from some literature). This means: 1) the N-glycans from insect cells are likely susceptible to Endo H treatment, which is great; 2) the N-glycans are not charged; 3) the N-glcans themselves are more or less homogenous as they are likely to be Man3,4,5 only, and are also smaller than the complex type glycans made from mammalian cells. This is a bless for crystallization without deglycosylation. Insect-made glycoproteins are still Endo H resistant. Commonly forgotten, they have (heterogeneous) core fucosylation at not only 6 but also 3 position of the innermost NAG. Endo H resistance can be remedied by addition of certain mannosidase inhibitors to your culture. Yeast-made N-glycans are, on the other hand, high-mannose and theoretically Endo-H sensitive (I have no personal empirical evidence). Engin
