Hi Sudheer, 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. Don't forget to multiply the value of C that you get by the protein weight in Daltons (calculated with the online tool as well) to get the mg/mL concentration and not the molar concentration if you prefer. For example, protein A has a MW of 21000 Da. The sample of protein A has shown an absorbance of 2.7 on the Nanodrop. Its extinction coefficient is known as 55000. The pathlength is 1. Therefore C (in mg/mL) is equal to (2.7/55000) x 21000. Regards toufic el arnaout ****************************************************** Toufic El Arnaout Membrane structural and functional biology Group Room 5.61 Trinity Biomedical Science Institute (TCD) 152-160 Pearse Street, Dublin 2 ****************************************************** 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
