Freimoser:Protocols:ProteinQuantification: Difference between revisions

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===Source===
The quantification methods listed below represent those that we find most useful. The descriptions are part of a more detailed list of protein quantification methods [http://dwb.unl.edu/Teacher/NSF/C10/C10Links/www.ruf.rice.edu/~bioslabs/methods/protein/protein.html]<br>
General Reference: Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990). [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B7CV2-4B5XNYK-1Y8&_user=791130&_handle=V-WA-A-W-ZZ-MsSAYWA-UUW-U-AAZVDYAEBB-AAZWBZWDBB-WVEVBEBE-ZZ-U&_fmt=full&_coverDate=12%2F31%2F1990&_rdoc=6&_orig=browse&_srch=%23toc%2318066%231990%23998179999%23473653!&_cdi=18066&view=c&_acct=C000043379&_version=1&_urlVersion=0&_userid=791130&md5=2124740cecad6e53b521353d92a0958c]
==Absorbance assays==
==Absorbance assays==


====Absorbance at 280 nm====
====Absorbance at 280 nm====
*Range: 20 micrograms to 3 mg
*Range: 20 μg to 3 mg
*Volume: Depends on cuvette - volumes range from 200 microliters to 3 ml or greater
*Volume: Depends on cuvette - volumes range from 200 microliters to 3 ml or greater
*Accuracy: Fair
*Accuracy: Fair
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====Absorbance at 205 nm====
====Absorbance at 205 nm====
*Range: Roughly 1 to 100 micrograms
*Range: Roughly 1 to 100 μg
*Volume: Depends on cuvette - volumes range from 200 microliters to 3 ml or greater
*Volume: Depends on cuvette - volumes range from 200 microliters to 3 ml or greater
*Accuracy: Fair
*Accuracy: Fair
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*Major interfering agents: Detergents, nucleic acids, particulates, lipid droplets
*Major interfering agents: Detergents, nucleic acids, particulates, lipid droplets


====Extinction Coefficient====
*Range: 20 micrograms to 3 mg
*Volume: Depends on cuvette - volumes range from 200 microliters to 3 ml or greater
*Accuracy: ~2% (very good)
*Convenience: Very good
*Major interfering agents: Detergents, nucleic acids, particulates, lipid droplets


==Colorimetric assays==
==Colorimetric assays==


====Modified Lowry====
====Modified Lowry====
*Range: 2 to 100 micrograms
*Range: 2 to 100 μg
*Volume: 1 ml (scale up for larger cuvettes)
*Volume: 1 ml (scale up for larger cuvettes)
*Accuracy: Good
*Accuracy: Good
*Convenience: Fair
*Convenience: Fair
*Major interfering agents: Strong acids, ammonium sulfate
*Major interfering agents: Strong acids, ammonium sulfate
====Biuret====
*Range: 1 to 10 mg
*Volume: 5 ml (scale down for smaller cuvettes)
*Accuracy: Good
*Convenience: Good
*Major interfering agents: Ammonium salts


====Bradford assay====
====Bradford assay====
*Range: 1 to 20 micrograms (micro assay); 20 to 200micrograms (macro assay)
*Range: 1 to 20 μg (micro assay); 20 to 200 μg (macro assay)
*Volume: 1 ml (micro); 5.5 ml (macro)
*Volume: 1 ml (micro); 5.5 ml (macro)
*Accuracy: Good
*Accuracy: Good
*Convenience: Excellent
*Convenience: Excellent
*Major interfering agents: None
*Major interfering agents: None
====Bicinchoninic Acid (Smith)====
*Range: 0.2 to 50 micrograms
*Volume: 1 ml (scale up for larger cuvettes)
*Accuracy: Good
*Convenience: Good
*Major interfering agents: Strong acids, ammonium sulfate, lipids
====Amido Black method====
*Range: 2 to 24 micrograms
*Volume: 2 ml
*Accuracy: Good
*Convenience: Poor
*Major interfering agents: None reported
====Colloidal Gold====
*Range: 20 to 640 nanograms
*Volume: 1 ml (scale up for larger cuvettes)
*Accuracy: Fair
*Convenience: Poor
*Major interfering agents: Strong bases

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Source

The quantification methods listed below represent those that we find most useful. The descriptions are part of a more detailed list of protein quantification methods [1]
General Reference: Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990). [2]


Absorbance assays

Absorbance at 280 nm

  • Range: 20 μg to 3 mg
  • Volume: Depends on cuvette - volumes range from 200 microliters to 3 ml or greater
  • Accuracy: Fair
  • Convenience: Excellent, if equipment available
  • Major interfering agents: Detergents, nucleic acids, particulates, lipid droplets

Absorbance at 205 nm

  • Range: Roughly 1 to 100 μg
  • Volume: Depends on cuvette - volumes range from 200 microliters to 3 ml or greater
  • Accuracy: Fair
  • Convenience: Very good
  • Major interfering agents: Detergents, nucleic acids, particulates, lipid droplets


Colorimetric assays

Modified Lowry

  • Range: 2 to 100 μg
  • Volume: 1 ml (scale up for larger cuvettes)
  • Accuracy: Good
  • Convenience: Fair
  • Major interfering agents: Strong acids, ammonium sulfate

Bradford assay

  • Range: 1 to 20 μg (micro assay); 20 to 200 μg (macro assay)
  • Volume: 1 ml (micro); 5.5 ml (macro)
  • Accuracy: Good
  • Convenience: Excellent
  • Major interfering agents: None
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