Hi Sudheer, As Dr. Irit answered. I would like to add that the Nanodrop actually gives the absorbance and not the concentration, but shows the absorbance units as mg/mL concentrations. So if you read 2.7 (absorbance units, AU), the Nanodrop will show it as 2.7 mg/mL. If I remember,the pathlength of the Nanodrop for the Beer-Lambert law is 1 mm, but the software shows the calculation converted for 10 mm (1 cm, therefore l = 1). You can submit the protein sequence to this online tool: http://web.expasy.org/protparam/ It will shows you the extinction coefficient, and the expected abosrbance for a 1 mg/mL solution of your protein.
Again, for your experiment, if you read on the Nanodrop for example an absorbance of 5, you need to use A=C.e.l l is 1, A is 5, and after you get e (ext. coeff.) from the online tool, you can calculate the actual C for your protein. Regards toufic el arnaout Today's Topics: > > 1. protein extinction coefficient (Sudheer Sangeetham) > 2. Re: protein extinction coefficient (Irit Rappley) > > > ---------------------------------------------------------------------- > > Message: 1 > Date: Wed, 30 Jan 2013 18:33:17 +0100 > From: Sudheer Sangeetham <[email protected]> > Subject: protein extinction coefficient > To: [email protected] > Message-ID: > <CAAdHTim= > [email protected]> > Content-Type: text/plain; charset=ISO-8859-1 > > Hello everyone > > I wanted to check my protein concentration based on its extinction > coefficient by using nanodrop rather than doing Bradford or Lowry methods. > I was checking the article in one article, The protein concentrations were > estimated using the extinction coefficient of 2.70 at 280 nm for a 1mg/ml > solution, but to the same protein in another article they determined the > concentration by giving 66350 /m/cm. If i want to check my protein > concentration by using nanodrop which value I should concern and how did > they get value of 2.7 ??? > > if anyone has idea please reply me > > Thanking you in advance > > Cheers > > -- > Sudheer Babu.S > Research Fellow > Institute of Biochemistry > Biological Research Center > Szeged,Hungary. > > > ------------------------------ > > Message: 2 > Date: Wed, 30 Jan 2013 11:36:13 -0800 > From: Irit Rappley <[email protected]> > Subject: Re: protein extinction coefficient > To: Sudheer Sangeetham <[email protected]> > Cc: "[email protected]" <[email protected]> > Message-ID: <[email protected]> > Content-Type: text/plain; charset="iso-8859-1" > > Hi Sudheer, > > The units for the extinction coefficient are 1/(M*cm), which match the > second value you mention. I would use that number. The calculation is (OD > 280nm) / (extinction coefficient). This will give you a concentration in > moles. You can multiply by 10^6 to get the concentration in micromoles, > which is more likely to be in the right range for your protein. > > Cheers, > Irit > > > > On Jan 30, 2013, at 9:33 AM, Sudheer Sangeetham wrote: > > > Hello everyone > > > > I wanted to check my protein concentration based on its extinction > > coefficient by using nanodrop rather than doing Bradford or Lowry > methods. > > I was checking the article in one article, The protein concentrations > were > > estimated using the extinction coefficient of 2.70 at 280 nm for a 1mg/ml > > solution, but to the same protein in another article they determined the > > concentration by giving 66350 /m/cm. If i want to check my protein > > concentration by using nanodrop which value I should concern and how did > > they get value of 2.7 ??? > > > > if anyone has idea please reply me > > > > Thanking you in advance > > > > Cheers > > > > -- > > Sudheer Babu.S > > Research Fellow > > Institute of Biochemistry > > Biological Research Center > > Szeged,Hungary. > > _______________________________________________ > > Methods mailing list > > [email protected] > > http://www.bio.net/biomail/listinfo/methods > > > > > ------------------------------ > > _______________________________________________ > Methods mailing list > [email protected] > http://www.bio.net/biomail/listinfo/methods > > End of Methods Digest, Vol 92, Issue 4 > ************************************** > > -- ****************************************************** Toufic El Arnaout Membrane structural and functional biology Group Room 5.61 Trinity Biomedical Science Institute (TCD) 152-160 Pearse Street, Dublin 2 ****************************************************** _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
