Common standard
* Standard curves are not always linear
* Standard curves may be linear over a small range
* The protein used for your standard curve must make sense
* Make sure your standard curve covers the absorbance range of your unknown with at least two points on either side
* If buffers or other contaminants are present in your protein preparations, use buffer blanks and a similar buffer system in your standards
* Make sure that your protein solution behaves in a reproducible manner to the assay method by making a dilution curve
* Use buffer and water blanks to anchor down your standard curve
* Place the protein concentration on the y-axis of you standard curve plot so that you can use the best-fit equation directly for concentration determination
* Use the amount of protein (i.e. micrograms) instead of concentration (i.e. micrograms per milliliter) to avoid confusion or mistakes
Two things must be understood about standard curves. First, they are called standard curves because the response of absorbance to the amount of added protein is normally not exactly linear or may be quite different from linear. Don't be too quick to use a program to fit a least-squares straight line to the data from your standard curve. Second, the range of protein you include in your standard curve (e.g. 0 to 25 µg or 0 to 100 µg) will depend on the assay being used. The ultraviolet methods are normally the least sensitive and require the highest amounts be included in the standard curve, whereas the Bradford method is sensitive, but not linear over a very wide range, necessitating the use of a relatively narrow range of standard protein.
Another thing to keep in mind is that your standard curve is based on one protein (usually BSA). The mixture of proteins you are assaying may not directly correspond to the standard you have chosen. While BSA in general is a good choice as a standard it does have limitations as a standard and this should be kept in mind when assaying your protein.
If your protein preparation contains buffers, salts or other contaminants you should consider using the same buffers, salts, etc. in your standard curve. This is very important when you are assaying a protein solution for the first time, because the presence of these components may have unforeseen effects on the assay.
Another thing to consider when assaying a protein mixture for the first time (especially in the presence of buffers and other contaminants) is to perform a dilution assay. If your dilution assay of your protein behaves in a manner similar to the standard you are using then you are fairly well assured that the standard you are using is appropriate. Doing a wide range dilution of your protein mixture during the assay as an internal control will accomplish this.
You also need to make sure that the unknown you are determining falls within your standard curve. It is usually desirable to have at least two points on either side of the determination of your unknown, and the more points on either side of your determination, the more accurate your results will be.
One small piece of advice with standard curves it to always place the protein concentration value on the y-axis of the curve. This allows you to directly use the best-fit equation for determination of protein concentration. It is also usually much easier to use the actual amount of protein as opposed to using concentration. This will eliminate errors that can be introduced by the utilization of different volumes of samples. For more information on this go here.
Finally, a buffer blank should be used. If you want to make sure that you can simply use a water blank you can run these blanks simultaneously and see how much they deviate from one another. The buffer blank should be used on all first time assays, because there is often a chance that the buffer you are using will interfere with the assay in some small way. Also, do not be duped by the instruction manuals that come with most protein assay kits. They will often say that certain buffers and salts do not interfere, when they mean to say that these reagents do not significantly interfere in the tests they have performed. To make sure that you are not introducing a contaminating substance into your assay the buffer blank is the best way to confirm this. This is also an easy and simple addition to any assay and will alleviate later headaches if done each time.
* Standard curves may be linear over a small range
* The protein used for your standard curve must make sense
* Make sure your standard curve covers the absorbance range of your unknown with at least two points on either side
* If buffers or other contaminants are present in your protein preparations, use buffer blanks and a similar buffer system in your standards
* Make sure that your protein solution behaves in a reproducible manner to the assay method by making a dilution curve
* Use buffer and water blanks to anchor down your standard curve
* Place the protein concentration on the y-axis of you standard curve plot so that you can use the best-fit equation directly for concentration determination
* Use the amount of protein (i.e. micrograms) instead of concentration (i.e. micrograms per milliliter) to avoid confusion or mistakes
Two things must be understood about standard curves. First, they are called standard curves because the response of absorbance to the amount of added protein is normally not exactly linear or may be quite different from linear. Don't be too quick to use a program to fit a least-squares straight line to the data from your standard curve. Second, the range of protein you include in your standard curve (e.g. 0 to 25 µg or 0 to 100 µg) will depend on the assay being used. The ultraviolet methods are normally the least sensitive and require the highest amounts be included in the standard curve, whereas the Bradford method is sensitive, but not linear over a very wide range, necessitating the use of a relatively narrow range of standard protein.
Another thing to keep in mind is that your standard curve is based on one protein (usually BSA). The mixture of proteins you are assaying may not directly correspond to the standard you have chosen. While BSA in general is a good choice as a standard it does have limitations as a standard and this should be kept in mind when assaying your protein.
If your protein preparation contains buffers, salts or other contaminants you should consider using the same buffers, salts, etc. in your standard curve. This is very important when you are assaying a protein solution for the first time, because the presence of these components may have unforeseen effects on the assay.
Another thing to consider when assaying a protein mixture for the first time (especially in the presence of buffers and other contaminants) is to perform a dilution assay. If your dilution assay of your protein behaves in a manner similar to the standard you are using then you are fairly well assured that the standard you are using is appropriate. Doing a wide range dilution of your protein mixture during the assay as an internal control will accomplish this.
You also need to make sure that the unknown you are determining falls within your standard curve. It is usually desirable to have at least two points on either side of the determination of your unknown, and the more points on either side of your determination, the more accurate your results will be.
One small piece of advice with standard curves it to always place the protein concentration value on the y-axis of the curve. This allows you to directly use the best-fit equation for determination of protein concentration. It is also usually much easier to use the actual amount of protein as opposed to using concentration. This will eliminate errors that can be introduced by the utilization of different volumes of samples. For more information on this go here.
Finally, a buffer blank should be used. If you want to make sure that you can simply use a water blank you can run these blanks simultaneously and see how much they deviate from one another. The buffer blank should be used on all first time assays, because there is often a chance that the buffer you are using will interfere with the assay in some small way. Also, do not be duped by the instruction manuals that come with most protein assay kits. They will often say that certain buffers and salts do not interfere, when they mean to say that these reagents do not significantly interfere in the tests they have performed. To make sure that you are not introducing a contaminating substance into your assay the buffer blank is the best way to confirm this. This is also an easy and simple addition to any assay and will alleviate later headaches if done each time.
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