dear everybody! i am very confused about this area and hope that you can help me out of this. for example this protocol: 1. Add sufficient bovine gamma globulin to each of eight test tubes to give you a calibration curve. Suggested values are 0, 10, 20, 30, 60, 90, 120 and 150 µg/ul protein. 2. Prepare 1:10 and 1:100 dilutions of the crude TDH prior to assay.Place 20, 40 and 60 µL of each TDH solution in separate test tubes. 3. Add water to a total volume of 150 µL in all tubes. 4. Add 5 mL of diluted dye reagent to each tube and vortex (carefully). 5. Incubate at room temperature for 5 minutes. 6. Transfer 200 µL from each sample and calibrator to duplicate wells on a microplate. and from this we read the OD and make the calibration curve. my questions are: 1. both the standard and sample solutions are diluted when we add water and dye reagent. this means that the standard solutions do change from their standard concentrations ( 0, 10, 20, 30, 60, 90, 120 and 150 µg/ul ). how come i use this calibration curve to measure the sample concentrations when the standard solutions are further diluted than suggested? and should i take the dilution factor from water and dye reagent into account when i find the real sample protein concentrations or are the values i get from the curve my real sample concentrations? 2. how can i make a standard curve using excel? does the computer automatically make it for us or should we do it ourselves? if so, then i hope that you can help me with it. thank you very much.