measuring protein concentration

[question]

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.

[kixxer]

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?

 

well, don´t take my word for it, but the way I understand it, the dilution with water and dye reagent do not matter in the measurement, because you use the same amounts of them on both the standard and the sample. So both OD´s you get are relative to each other. The reason why you dilute the samples anyway is to get to a OD-region which is nicely measurable, if there is a too high protein concentration in the tube, the test will produce wrong numbers. In the end all you do is compare the OD you got from your sample with the one of your standard. And you only can compare them if they are equally diluted.

 

But as I told you, I´m not the expert on this stuff either:(., so this stuff might be horrible wrong too. Some experts help here please?

 

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.

 

you can make a Chart with your datapoints (OD´s of the standard) and then let Excel add a trendline to the chart. For this, you need to right-click on the graph and choose "add trendline --> linear".

 

I hope I could help you a bit,

 

greets,

kixxer

 

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