This is my first time trying Western Blotting and I need help with managing the unknown protein quantification using a plate reader (SpectraMax/Softmax Pro software).
I have an African cichlid brain sample to which I have added 150μl of diluted PBS buffer (PBS+ Phosphatase/Protease inhibitors+mild detergent)
I was told I need to do a serial dilution before I can add the Bradford Reagent.
In terms of the standard curve, I am using 1 mg/mL BSA stock with 1, 0.5 , 0.25 , 0.125, 0.063, 0.031, 0.016 and 0 mg/mL BSA dilutions; 10 μl from each dilution and 160 μl Bradford reagent.
I started with 600μl of BSA and 300μl of distilled water when diluting for the standard curve, but I do not have as much of the unknown sample and I need to make sure not to use up a lot of my sample so I can run the gel.
How do I do serial dilution with 150μl of unknown protein?
Is there a better protocol to quantify protein with the plate reader?
If anyone could help with this, I would appreciate it immensely.
First, concerning your standard curve, 2-fold serial dilutions are not ideal for this purpose. The useful protein concentration range of the Bradford assay is pretty narrow. I suggest you use arithmetic (evenly-spaced) dilutions. The correct range depends on the quality of your Bradford reagent. With the one from Bio-Rad, which is very sensitive, I would use between 0 and 5 µg of BSA in 10 µl of buffer, in 0.5 µg increments, + 200 µl of Bradford reagent.
Second, detergents can interfere with the Bradford assay. Check this document to see if the detergent you are using will interfere. If it will, switch to a different method, such as the BCA assay.
https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6852.pdf
Before running the full assay on your sample, I suggest you to a small test run to get in the right range. In a part of the plate you don't need, add 1, 2,4 or 8 µl of sample, equalizing the amount of the buffer the sample is in, one well of each, then 200 µl of Bradford reagent. Once you have figured out the right amount of sample to be somewhere in the middle of the standard curve, make a fresh measurement in triplicate of just that dilution. That will save a lot of sample.
Since the sample contains detergent, you will have to include the detergent in the standards as well.
Dear Prof Adam B Shapiro
Can different buffers give different results. In other words, if we used two different diluents (both of them reported as an appropriate diluents in the kit's manual), would the concentrations be different in this case? of note, standards and samples were diluted in the same diluent.
Thank you in advance.
If the diluent contains something that causes interference in the assay, it might cause different results from a diluent that does not interfere. But in most cases, I would expect to get the same result for different diluents as long as standards and samples are in the same diluent, and the concentration of the diluent is the same for all standards and samples.
When I dilute samples for the Bradford assay, I normally just use distilled water.
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