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.
The value they gave you should be the final value after you add water to 150 uL. The reagent will not have an impact on the OD value and its not view as dilutent in this case. The value you get from the curve should be the estimated concentration of you unknown samples tubes. To find the real value of you unknown sample you will have to take into account the volume added and the dilution. You have to a scatter/point graph then have "linear curve fit" and select "show formula" for the curve fit. All you have to do is plug y (OD) and solve for x (concentration).
so we always ignore the volume of the dye reagent. i was very confused, because sometimes we have to add 1ml of this dye and practically it should dilute the concentrations, but then it does not. you mean the water volume and also the dye volume? or just the water volume? i will try and hope that it works. but does it mean that we can always make the curves from a normal excel or does it need specialized program for making it? thank you very much!
hi Ian, i could not make it. my excel is from Microsoft 2002. i don't know how i can get the menu "linear curve fit". where does it lay? is it in the excel sheet or in the dialog box which appears? hope for the reply. thanks.
Yes, it is the sample that will have an impact on the OD value because it reacting with the dye. More or less dye will have an impact on the OD but not on the concentration. the volume of the unknown sample, 20 or 40 or 60. The protein concentration in the measured sample will be different but it should be linear and give you the same concentration for the unknown sample. http://www.owlnet.rice.edu/~chem121/lab/pennies/graphhelp.html
it seems like my excel does not have Trendline menu. is this something we automatically get when we have the excel or should i have to install it?
It usually in the program. Maybe this will help http://office.microsoft.com/en-us/assistance/HP052069791033.aspx
As with most assays of this type, you aren't directly determining concentration, you are determining the amount of protein (or whatever else you're interested in measuring) in the solution. You then calculate the concentration based on the amount of sample and any dilution factors if you had to dilute your sample relative to the standards in order to get an amount of your protein that was measurable on the standard curve you have.
thanks all for the help. i am still not sure how to calculate my protein concentration. i hope you can give me some advices with these problems: first problem: 1. dilute your samples 5X. 2. add 25ul of your diluted samples and the standards into separate tubes. 3. add 125ul reagent I into each tube 4. add 125ul reagent II into each tube. 5. discarded all supernatants in all tubes. 6. add 127ul reagent A into each tube. 7. add 1ml reagent B into each tube. 8. read the OD after 15min incubation. If the standard equation is: y=0.2622x and one of my samples has an OD of 0.266. from the equation my sample would be: x= 0.266/0.2622, x= 1mg/ml. this is the concentration of my sample from the curve, but what is the real concentration? Should I also multiply with the dilution factor, 5? I am confused because even if we dilute the sample 5X, but we do discard (throw away) all the supernatant. In this case the sample gets concentrated and not diluted . hope for tips. Second problem: • add 20 µl of undiluted samples and standards into individual wells of a 96-well plate. • Add 40 ul of Bradford Reagent into all wells containing standard or sample. • Add dd H2O to all wells to bring the final volume to 200 µl. • Read absorbance at 595 nm without any prior incubation. If the standard equation is: y=0.2622x and one of my samples has an OD of 0.266. how can I calculate my sample concentration? The sample is undiluted but then we add 140ul dH2O into both the undiluted samples and standards. Should I use this 140ul volume in my calculation or should i ignore it since it is also added to the standards? thanks!
Reagent I and II probably precipitate your protein. So the amout of protein does not change and it will not influence the concentration because the standard go through the same process. It will depend on the way the protein standard concentrion is set but most of the time to calculate the concentration, you mutiple the x value by the dilution factor. It will depend on the way the protein standard concentraion was done but usually you take the x-value as the concentatrion of protein solution.