Originally Posted by danfive
You read absorbance on a spectrophotometer.
Calculate average absorbance for each std and sample. Use the avg absorbance for calculations.
Plot Absorbance values of standards only (x-axis) vs Protein conc (y-axis).
Extrapolate the linear or quadratic (best fit) line(and equation) for Abs values of the standards.
Use the formula for std curve to calculate the protein conc of samples/unknowns.
You do this by substituting Avg Absorbance (of sample) as x into the linear/quadratic formula; y =protein conc, x = avg absorbance.
I do this easily on excel, some spectrophotometers have built-in programs.
Like I mention in the above post, I do this on Excel--the spreadsheet program. I do this because the thermo spectrophotometer I use has horrible software on it.
Anyways, although, it is common sense to put the independent variable--protein conc on the x-axis, and the dependent variable (Abs) on the y-axis. I do it my way,
because it makes no difference, and I get excel to solve for x (protein conc) by flipping the axes. That is by switching the x and y values, excel, which only solves for y, solves the equation for protein conc.
I can prove that it makes no difference, because I can just as easily switch the axes just as the last two posters mentioned. Resulting in an equation that requires me to manually solve for x. Solving for x, again gives me the same equation as flipping the axes.
In summation, following the advice of the last two posts, I get this:
Abs=Protein conc*constant + y-intercept
*this requires I manipulate and solve for Protein conc. I have Abs, I need Protein conc.
My way, w/ axes switched, gives this:
Protein conc=Abs*constant + y-intercept
**Excel program solves it for me!