Standard curve

[Jimmy B]

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Does anybody know a good website that explains this? I have to do one for glycogen-the concentration is 20mg/ml of 100 microliters. I diluted this to 5mg/ml. The amount of glycogen one study used was in the nmol amount. Another study did the curve starting at 10 micrograms of glycogen. Bascially I am trying to figure out how exactly I'm going to do this and how I will plot the curve (how much glycogen will I begin with-like 2micrograms or something).

 

[RxnMan]

The only "standard curve" I've ever heard of is where you use a set of serial dilutions (1:10, 1:50, 1:100) of a chemical (say CuSO4) and test them against some instrument (Spect-20) and plot each response against dilution. You won't get a curve per-se - it's almost always a straight line - but you can use this chart to determine the unknown concentration of a test sample of CuSO4 solution (if the test solution doesn't fit on your curve, dilute it down, keep track of your dilution factors, and work backwards).

If you're having trouble finding a place to start, first try to get the two papers into similar units. Maybe something can shake out there and give you a place to start. Otherwise, just copy one paper's methods and get to it. Often the reasoning behind a group's methods becomes apparent after you've gone through the protocol a few times. Hope this helps.

 

[Jimmy B]

That helps a little bit. My only concern is whether or not I will have the right amount of glycogen when I diulte it. For instance, I'm starting with 20mg/ml of glycogen and my PI said that 20mg/ml=20microg/microL (which is the concentration that the paper used) but they plotted micrograms of glycogen(not the concentration) versus the Abs. This is where I am stumped.

 

[RxnMan]

Then they assumed units when they wrote the figure (not good practice, but they may have been restricted by space). Look at the figure context (caption + when it is referenced in the body of the article) to see what units they're talking about there. From everything else you've said, they probably (but do not take my word for it and check for yourself) used microliters.

Did you try to convert the data in the other paper from nmol/microL => microgram/microliter?

In any case, you should make a run of dilutions starting with 20 micrograms/microliter and see how it goes. If your slope is roughly the same as the paper's, then you're probably golden.

 

[Jimmy B]

Here's what's in the paper in reference to their figure:

"Triplicate samples of standard glycogen solutions containing from 5 to 100 micrograms of glycogen were subjected to steps 8-13 (their protocol). The average readings of their absorbance were plotted against the amount of glycogen used to produce a standard curve. The mean slope calculated from the 33 glycogen standard samples ranging from 5 to 100 micrograms was 0.0101..."

It seems to be a bit more involved from what I'm trying to do (they used 33 samples-I'm using 5 in a serial dilution).