Warm up the spectrophotometer. Switch it on and leave it on for at least 15 minutes before use.
Prepare "standards" in the assay plate. Standards are solutions containing known amounts of the glycoprotein for which you will be testing your experimental compound. Obtaining absorbance readings for these known standards, and subsequently a calibration curve that plots known concentration versus absorbance, will enable you to quantify the amount of protein in your unknown samples.
To prepare your standards, fill all 12 wells in a row of an assay plate with 100 microliters distilled water, using a micropipette. To the first well, add 100 micrograms of glycoprotein by pipetting. Use a micropipette to thoroughly mix the glycoprotein and distilled water together: pipette up and down. This first, most concentrated well contains a ratio of 50 micrograms of glycoprotein to 50 micrograms water. Remove 100 microliters of mixed liquid from that first well and pipette into the second well. Mix thoroughly by pipetting up and down. The concentration of this second well is 25 micrograms glycoprotein to 50 micrograms of water. Remove 100 microliters from the second well, pipette into the third well and repeat the procedure for all the subsequent wells in the row until you reach the last well. The last well should consist only of distilled water and have 0 micrograms of glycoprotein, so do not pipette into the last well.
In other words, each of the standard wells should have 100 microliters of fluid in them and should decrease, in glycoprotein content, by half each time, from 50 micrograms to 0 micrograms.
Place 100 microliters of your unknown compound into an empty well of your assay plate. You do not need to dilute the unknown well, because you do not need a calibration curve from the unknown. Therefore, each unknown that you are testing for glycoproteins only requires one well in the assay plate. Your unknown should contain an estimate of between 5 and 100 micrograms of glycoprotein. This estimate increases the likelihood that the absorbance readings of your unknown will fall on the calibration curve that you obtain from your standards.
Add dye and reagent, in quantities specified by the reagent kit manufacturer, to the standards and unknowns. Wait five minutes to allow for formation of complex.
Read the absorbance of both the standards and the unknowns in the spectrophotometer. From the standards, obtain a curve calibrating absorbance values versus known concentration. The spectrophotometer should automatically formulate this for you, but if not, make your own curve. Using a graphing program --- or graphing paper --- plot absorbance values on the x-axis and known concentration on the y-axis. Fit a best-fit line to the calibration curve to obtain your relationship between absorbance and concentration.
Plug your raw absorbance values for the unknown samples into your calibration curve formula, to obtain the concentration of glycoprotein in your sample.