Quantification by Absorbance at 280nm: Difference between revisions
wrote initial page |
No edit summary |
||
| Line 3: | Line 3: | ||
#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]] | ||