Quantification by Absorbance at 280nm: Difference between revisions
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#Using nanodrop measure absorbance in triplicate, ensuring to blank against an appropriate buffer. | #Using nanodrop measure absorbance in triplicate, ensuring to blank against an appropriate buffer. | ||
#Calculate extinction coefficient for protein of interest | #Calculate extinction coefficient for protein of interest | ||
:#Use the [http://www.expasy.ch/tools/protparam.html | :#Use the [http://www.expasy.ch/tools/protparam.html Protparam] tool to calculate the extinction coefficient in both molar and mg/mL for protein. Assume all Cys residues are half-cysteines. | ||
:#If the protein is a GST-fusion add 43110 M<sup>-1</sup> and 1.607 mg/mL<sup>-1</sup> respectively | :#If the protein is a GST-fusion add 43110 M<sup>-1</sup> and 1.607 mg/mL<sup>-1</sup> respectively | ||
#Divide the absorbance value (normalized by dilution factor if necessary) by the extinction coefficient | #Divide the absorbance value (normalized by dilution factor if necessary) by the extinction coefficient | ||
#If necessary adjust by the percent purity as described in [[Determining Percent Purity]] | #If necessary adjust by the percent purity as described in [[Determining Percent Purity]] | ||
Latest revision as of 14:03, 11 May 2009
This method is most accurate with highly purified proteins.
- Using nanodrop measure absorbance in triplicate, ensuring to blank against an appropriate buffer.
- Calculate extinction coefficient for protein of interest
- Use the Protparam tool to calculate the extinction coefficient in both molar and mg/mL for protein. Assume all Cys residues are half-cysteines.
- If the protein is a GST-fusion add 43110 M-1 and 1.607 mg/mL-1 respectively
- Divide the absorbance value (normalized by dilution factor if necessary) by the extinction coefficient
- If necessary adjust by the percent purity as described in Determining Percent Purity