Bio-Rad Experion Protein Analysis Kits - Manual (Page 22)

Notes: The Experion system uses a single-point calibration to the upper marker protein for
relative quantitation; therefore, relative quantitation accuracy is often highest for proteins
that stain with similar efficiency to the upper marker protein. Though the accuracy of this
method for some proteins can be high (within <0.5% of expected)
4 , accuracy for other
proteins, such as the BGG standard protein, may not be as good. Such differences in
quantitation accuracy for different proteins also occur with other commonly used protein
quantitation methods, such as the Bradford and Lowry assays
5–8 , and are often due to
differences in protein staining efficiency; unique protein characteristics can affect the levels
of staining and colorimetric signals produced.
Because proteins exhibit differences in staining behavior, alternative protocols exist for their
quantitation: relative quantitation against a user-defined internal standard and absolute
quantitation. Perform Test 3 to see how absolute quantitation improves quantitation accuracy
for the BGG standard. For more information on the quantitation methods used by Experion
software, refer to Appendix A.
If your results do not appear as they do in these examples, refer to the troubleshooting
section of the Experion Pro260 analysis kit and Experion system instruction manuals.
Typically, problems with reproducibility are due to problems with pipetting or chip loading.
For tips on optimizing your results, refer to the Experion Training Video: Chip Loading and
the Essential Practices and Troubleshooting sections of the Experion system instruction
manual.
4 Nguyen M and Strong W, Performance comparison of the Experion automated electrophoresis system and a competing
automated system for protein analysis, Bio-Rad bulletin 5302 (2005)
5 Bio-Rad bulletin 1069, Colorimetric protein assays
6 Bradford MM, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of
protein-dye binding, Anal Biochem 72, 248–254 (1976)
7 Lowry OH et al., Protein measurement with the Folin reagent, J Biol Chem 193, 265–275 (1951)
8 Wu F and Strong W, Comparison of protein quantitation methods using the Experion automated electrophoresis system,
BioRadiations 123, 28–31 (2007)
18
10010510A.qxp 2/13/2008 11:43 AM Page 18