Using a Pierce BCA Assay kit for whole protein quantification, our lower end of the curve is not sensitive to picking up even our standards for 25 µg/mL. We are using microplates. Does anyone have experience with this problem, and how did you fix it?
Are there other assays to consider that are both sensitive and accurate for lower-end concentrations of whole protein? We are trying to avoid using fluorescence based assays if possible. Thanks in advance!
Hi Melissa,
for total protein quantification we use quantitative amino acid analysis. About 2-5 us total protein is needed, the sample can be speed vac to load suffient amounts. This is the most accurate method to quantitate total protein or a purified protein. See more about analysis principles and sample prep at www.Alphalyse.com
In the microplate protocol of the BCA assay, the sample volume is up to 25 µl. Therefore, your 25 µg/ml standard contains 0.625 µg of protein, which is at the lower limit of sensitivity of the assay, assuming no interfering substances are present. Make sure you warm the plate as instructed to maximize the color development.
If you have very dilute samples, and a large volume of them, you can concentrate the samples to a smaller volume to increase the protein concentration. This can be done non-destructively using ultrafiltration, or destructively using precipitation with trichloroacetic acid. In the latter case, the protein pellet can be dissolved directly in the assay reagent. The sample can then be transferred to the microplate for measurement.
Generation of the 562nm signal in the BCA assay is dependent on time and temperature. It has been my experience that when quantifying low concentration samples it helps to heat the assay at a higher temperature and let the assay go for 90-120 minutes. For the elevated temperature I seal the 96-well plate with parafilm (to prevent evaporation), put the plate in a plastic sandwich box and place the setup in the 55C agar tempering water bath. Keep in mind that the signal development will eventually reach completion so some of your high standards will fall out of the linear range (you can adjust your standard curve to a lower concentration range). I would also second the previous comments about being wary of interfering substances in your sample- this includes the usual suspects of imidazole, EDTA, DTT, BME, citrate and reducing sugars such as maltose. You can compensate for some of this interference by making your standards in the same buffer as your sample but you will have a dampened signal and lowered sensitivity (especially with chelators like EDTA and imidazole). If the working reagent immediately shifts to a blueish color when added you have a chelation problem and will need to dialyze/desalt/ultrafilter it out or try a different quantification system.
I'm not sure what you are doing with your sample but depending on the accuracy required you may be able to use the good-old Bradford assay, it would probably be good enough for quantifying crude protein concentrations for something like a normalizing loads for a Western. Commercially available versions are inexpensive, last for years in the fridge and are compatible with many common reagents that interfere with the BCA. There are make-it-yourself Bradford protocols using phosphoric acid and Coomassie dye but the preparations aren't storage-stable and can vary between batches and just aren't worth it in my opinion.
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