Re: [ccp4bb] how to quantitate protein which dont have ne aromatic residue
In general, most protein quantification methods have significant problems with idiosyncracy, because proteins are quite variable in composition and structure. The best method is to use is the absorption at 280 nm, but this is quantitatively useful only if the molar absorptivity is known (and the protein is pure, or at least purified away from UV-absorbing materials). We work extensively with metalloenzymes, and quantifying protein:metal ratios accurately is highly problematic. Here is my take: The Bradford (dye-binding) assay is quite dependent on protein composition (hydrophobic content and perhaps other amino acid composition). We see variances of apparent to actual protein concentration of 50% or more The Lowry-type assays based on molybdenum blue formation (this includes the BCA assay which is a variant of the classic Lowry assay) are also highly composition dependent. We see variances of apparent to acutal protein concentration of up to 100%, depending on the protein involved. The Biuret assay is dependent primarily on the peptide backbone content, and has small variation of response to composition. It is not very sensitive, however. The microbiuret assay, which is similar to biuret but with measurements made at 330 nm is tolerable. Protein must be homogeneous, of course. We see relatively little variation (20% or so) between apparent and true concentrations of protein using this method, but it is subject to a variety of interferences. Far UV absorption of proteins is nearly independent of composition, as absorption in this region is due almost entirely to peptide bonds. This requires purified protein, and the absence of other UV-absorbing materials, but is otherwise quite tolerant. Maximal consistency can be obtained by carrying out measurements in denaturing detergents. We have used 0.01-0.1% Triton X-100 with success. The asborptivity of 1mg/mL solutions is approximately 20.5 at 210 nm and around 34 at 205 nm. various corrections can be applied to correct for absorption of aromatics at 205 nm. See Scopes (1974) Anal Biochem 59, 277-282. The systematic variance of this method is generally small, maybe 20% or less. I have found that methods 3-4 frequently agree, and give good agreement with well-characterized metalloproteins where the metal can be very accurately measured using ICP-OES. Cheers. -- Roger S. Rowlett Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu
Re: [ccp4bb] how to quantitate protein which dont have ne aromatic residue
Hi Arpita You can try QUANTI-iT Protein assay kit from Invitrogen. But still there is nearly 20-50% discrepancy between this method and a Abosorbance at 280. I also faced same problem with a protein, then re-cloned by adding a Trp at the C-terminus. Raj E. Rajakumara Postdoctoral Fellow Strcutural Biology Program Memorial Sloan-Kettering Cancer Center New York-10021 NY 001 212 639 7986 (Lab) 001 917 674 6266 (Mobile) --- On Sat, 9/4/11, Arpit Mishra ar...@igib.in wrote: From: Arpit Mishra ar...@igib.in Subject: [ccp4bb] how to quantitate protein which dont have ne aromatic residue To: CCP4BB@JISCMAIL.AC.UK Date: Saturday, 9 April, 2011, 3:22 PM 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
Hi, You can read the spectrophotometric absorption at 280 nm and 200nm in UV range. It should serve your purpose and provide a decent idea for the amount of protein in the sample. Provided that absorbance at 280nm is given by aromatic rings but at the same time absorbance at 200nm is contributed by peptide bonds. A simultaneous reading at 260 nm which can be deducted later will substract the effect of nucleic acids. The relation between the readings at these values and protein concentration you can easily find anywhere on the web. Best wishes Gauri
[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
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
