Bio-Rad SmartSpec Plus Spectrophotometer - Manual (Page 21)

The SmartSpec Plus Bradford, Lowry, BCA and Other Protein assays use standard
curves created by measuring the absorbances of solutions of known concentration
to determine the concentration of unknown samples. If you do not want to construct
a new standard curve, you may recall one from the on-board memory. SmartSpec
Plus has the capability of storing up to ten standard curves in memory. The stored
curves are numbered automatically from 0 to 9, or you can overwrite the number.
Stored curves may also be assigned alphabetic names using the Alpha function of
the number pad.
You also have the option of declining construction or recall of a standard curve,
but without the standard curve, the SmartSpec Plus cannot convert absorbance
readings to concentrations.
The UV Protein assay, developed to estimate protein concentration in a nucleic
acid background, as in a crude cell extract, calculates protein concentration by the
equation
Concentration = C1 * (A
280
– A
320
) – C2 * (A
260
– A
320
)
where C1 and C2 are constants. If you select the UV Protein assay, SmartSpec
Plus will first show you the current values for C1 and C2 and ask you to accept or
modify them. The preset values for C1 and C2 are 1.55 and 0.76 respectively.
Standard Curve Construction. SmartSpec Plus will calculate a mean and
standard deviation for each standard replicate group (if replicates are specified)
and then use those data to fit a linear standard curve. The instrument will report
the slope and intercept of the standard curve and the square of the correlation
coefficient for the curve (r
2 ).
The correlation coefficient (r) provides an indication of how well the linear
polynomial regression model fits the data. In effect, the correlation coefficient
is the square root of the proportion of explained variation to total variation of
the regression.
r = (explained variation / total variation) 1/2
The amount of explained variation increases as the goodness of fit improves. In a
perfect fit, all of the variation is explained, the explained variation equals the total
variation, and the ratio of the explained variation to total variation becomes 1.000.
Or, more simply, the closer the correlation coefficient is to 1.000, the better the fit
of the regression, and the better the estimate of concentration.
Storing Standard Curves. As many as ten standard curves may be stored in
SmartSpec Plus’s on-board memory. These may be recalled for use in determining
concentrations in subsequent assays. A new curve will be assigned a number from
0 to 9 by the firmware and you can then give it an optional name of up to eight
characters in length.
Printing Standard Curves. If you choose to print a full report at the end of an
assay, the standard curve will be printed along with the information about the
curve. This will include the concentrations and absorbances of the standard and
the slope, intercept and coefficient of the curve.
You can choose to print the same information about a standard curve when it is
first created or when it is recalled from memory.
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