I am wanting to know whether it is important to plot standard with BSA again and again while doing the protein estimation if I am using a Bradford reagent from the same bottle (I use GeneITM kit of Bradford's estimation)? Does anybody have any suggestions?
This is an interesting issue...I would say, that the mean of 3 such determinations should be enough. The only reason to do this standard curve is to be able to determine your protein's concentration. But I think, that some poeple wouldn't agree with this.
I still believe that we need to make standards every time we run measurements. the reason is we can not sure that the concentration of all substances in the reagent are not changing during storage. Even though the concentration may change a bit, it might change the absorbance measured. The instrument you are using also may give slightly different response between one measurement and another. You may notice that you rarely (or even never) get the exact same linear regression of standards if you repeat your standards measurement between days. So, in my opinion the best practice is to run standards every time you measure your samples. However, I would not say its totally wrong to use a standard curve for a routine work, as long as you make sure that the error between one standard and another are not too high.
I agree with Safri for all the reasons stated above. It depends on what you are going to do with the protein. If you are doing an experiment in which concentration is small, for instance a GST Pull down or EMSA, difference in concentrations can be an important issue and the reason for which your experiment could not be reproducible. I do the plot standard every time; it’s not that time consuming.
Hi Prem,
Yes, the best thing is to make standards every time.
However, if you do not care about absolute concentration rather relative protein concentration (for example, if you need to compare your mutant protein amount to your wildtype control sample...) no need to do the standards every time. In fact this is what I do, when I have a new bottle of Bradford (diluted and filtered) I make a good standard curve and use this curve until the whole bottle is gone. Have been doing this for over five years and never had a problem so far.
Even, when I also agree with my previous speakers, you will always have these variations mentioned by Safri. That's why, a mean value with a low standard deviation is fully sufficient. Of course, you can do this everytime, it's not much work, but in my opinion it's not necessary.
Even with the same batch, if you do the experiment with Bradford reagent due to its highly viscous nature, there will be significant changes
while dye binding occurs. This changes the OD and the corresponding concentration of the protein in absence of a parallely run standard
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