Protocol Online logo
Top : Forum Archives: : Protein and Proteomics

measuring protein concentration - (Feb/27/2005 )

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 g/ul protein.
2. Prepare 1:10 and 1:100 dilutions of the crude TDH prior to assay.Place 20, 40 and 60 L of each TDH solution in separate test tubes.
3. Add water to a total volume of 150 L 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 L 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 g/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.

-indoubt-

hi!
I'll try to answer your questions:


QUOTE
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 g/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?


what you're measuring is in fact higher dillued than in the beginning, BUT you're comparing the same dillutions of sample and standard, so you can neglect all steps done to both of them.

example:

you use 25l standard with know concentartion of 1g/ml
and you use 25l of your unknown sample.

both are dilluted in the same steps
both give you the same OD reading at the end of the test, so
both have to be concentrated roughly the same.

So you know your sample contains 1g/ml protein.

I said roughly, since no protein detection test is ever exactly correct, since there are many factors that can give you slightly misleading results (e.g. using BSA as standard).

QUOTE
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.


Quite easy:

get your data organised in that way:



(known concentration) (measured value)

e.g.:
(1) (0.2)
(10) (2)
(100) (20)

(please remember that I'm a user of a non-English MS-Office, so exact terms may vary...)
put that into excel, mark the whole area, and press the button to make a graph. select "point" as graph type and press "finish". then go to your graph, left-click on one datapoint then right-click. select "add trend", use linear regression as type, click on options an mark "show equation". then you get a line drawn in your graph - that's the regression curve, and a equation like:


y = 0.2x - 2E-15


this equation you can use to caculate your samples protein concentration:

e.g.:

sample value: 1.9

x = (y - 2E-15) / 0.2

here:

x = (1.9 - 2E-15) / 0.2 = 9.5



Ok, I hope that helped.

mike

-jadefalcon-

thank very much mike!

when i calculate my original sample concentrations, should i ignore the dye volume or not?

i will try the graph and hope it works. biggrin.gif Thank you very much again.

-indoubt-

QUOTE
when i calculate my original sample concentrations, should i ignore the dye volume or not


As I said, all steps that are done to the sample as well as to the standard dillutions you use cancel themselves out in the calculation. So if you're adding the same amount of dye to your sample and your standard, you can ignore it.

mike

-jadefalcon-

thanks, mike!

but what happens if i add different volume of dyes into the samples and standard solutions? should i in this case use the dye volume in my calculation too? sad.gif

-indoubt-

QUOTE
but what happens if i add different volume of dyes into the samples and standard solutions? should i in this case use the dye volume in my calculation too?


As I understood, you said you added the same volumes to all the samples, including the standards (as it usual for all the protein detection kits I know):

QUOTE
4. Add 5 mL of diluted dye reagent to each tube and vortex (carefully).


IMHO, if you add different amounts of dye to samples and standards, you can't use the results anymore, since different dye concentrations will falsify your test....

mike

-jadefalcon-