The y-axis and Protein Concentration

-
Here is a hypothetical example that illustrates why it is more convenient to use the y-axis to place your protein concentration.  For a particular assay let us say that we get the following data.  We will organize it and make standard curve plots for both configurations

Case #1:  Protein on the x-axis

x-axis Values y-axis values
µg Protein A590
1 .2
2 .8
3 1.2
4 1.8
5 2.0
 We will get a standard curve that looks like this...
Standard curve example with protein on the x-axis
This standard curve has a curve fit line with the equation
y = 0.46*x - 0.18
Our x-values are µg of protein and our y-values are A590.  Thus, we can rewrite the equation to be more useful.
[A590] = 0.46*[µg Protein] - 0.18
The measurement we are taking is the absorbance of the sample, so in order to calculate the µg of protein in our unknown sample we have to rearrange the equation to be...
[µg Protein] = ([A590]-0.18)/0.46
While this is not a difficult rearrangement, it does require some time to do and could introduce errors into the calculation of the amount of protein.

Case #2:  Protein on the y-axis

Now let us take the case where we put the amount of protein on the y-axis of our standard curve with the same above data, now rearranged.
x-axis Values y-axis values
A590 µg Protein
.2 1
.8 2
1.2 3
1.8 4
2.0 5
We get the following standard curve...
Example of putting protein on the y-axis
This standard curve has a curve fit line with the equation
y = 2.1296*x + 0.4444
Our x-values this time is A590, whereas our y-values is µg Protein.  Thus, we can again rewrite our equation to read...
[µg Protein] = 2.1296*[A590] + 0.4444
This time we do not have to rearrange our equation before using it.
In the case where a spreadsheet is being used to handle standard curve and experimental data, the power of this become much more apparent.  When using a spreadsheet you can directly copy you line fit equation from your graph and paste it into your spreadsheet formula, thus simplifying the whole process of analyzing data.

Comments

Post a Comment

Popular posts from this blog

Prepare 6X Laemmli buffer

-
Laemmli is a sample buffer to use in western blot. 6X sample buffer is added to each protein sample and is boiled or heated for 5-10 minutes. Recipe to prepare 10 ml: - 1.2gr SDS (sodium dodecyl sulfate) - 6mg bromophenol blue - 4.7ml glycerol - 1.2ml Tris 0.5M pH6.8 - 2.1ml dwater warm it a little bit and shake it till everything is dissolved. It takes time. - add 0.93gr DTT. Wait until it is completely dissolved. Aliquote and keep frozen at-20ºC.
Read more

0.1 M Triethanolamine

-
0.1 M TEA: TEA = Triethanolamine You will dilute TEA 1:10 in Millipore water: Pour 50 mL TEA into a 50 mL conical tube (TEA is very viscuous, so best to measure it in something that is disposable). Pour TEA from conical tube into 450 mL Millipore water with stirring. pH to 8.0
Read more

Laboratory Maintenance Checklist

-
Image
It is a known fact for anyone working in a laboratory be it the students, the researchers, scientists or lab assistants, it is necessary to follow safety rules and guidelines for their own safety. All laboratories have their lab safety symbols and lab safety equipments like goggles, mask, dress etc. At the same time, it is also very essential that a laboratory is well-maintained and taken care of so as to prevent any kind of dangers involved while working in a lab. As per rule, there should be routine maintenance and safety checks in a laboratory. Infact, it should be the duty of every person working in a laboratory to do a quick safety check after the completion of each experiment and before the starting of a new one. Or a responsible personnel should be assigned the duty to clean the laboratory and perform simple maintenance for lab care and safety . Laboratory Maintenance Presenting below few points which should be considered for laboratory maintenance: Bench:
Read more