Bradford assay and BCA kit is unable to measure protein concentration due to the presence of some reducing sugars in the sample by giving high intensity color.
Precipitate the protein and wash the solid obtained. If precipitation cannot be performed, use dialysis or ultrafiltration to wash out the reducing sugar.
Thank you, Omar. I need to study release kinetics of the protein. Moreover, sample size was very high, but volume would be very less for dialysis or ultrafiltration.
The Bradford assay should not suffer interference from reducing sugars. It can tolerate up to 20% glucose. There may be something else in your samples that interferes with the Bradford assay, such as detergent. Here you can see the compatibility table for Bradford reagent.
https://www.bio-rad.com/webroot/web/pdf/lsr/literature/4110065A.pdf
Depending on what else is in your sample, and the released protein concentration, you may be able to follow protein release by 280 nm absorbance.
You can use the protein precipitation technique, as suggested by Omar Gonzalez-Ortega on 1-ml samples of the released protein solution usinf 1.5-ml microcentrifuge tubes for sample processing. The pellet can be washed if necessary, or if not, it can be used directly for the Bradford, BCA, or Lowry assay.
An example method is as follows:
To 1 ml of dilute protein solution in a 1.5-ml microcentrifuge tube, add 0.1 ml of a 0.15% (w/v) aqueous solution of sodium deoxycholate and 0.1 ml of a 72% (w/v) aqueous solution of trichloroacetic acid. Incubate 10 minutes on ice. Centrifuge at top speed in a microcentrifuge (e.g. 16,000 g) for 10 minutes. Very carefully remove all of the supernatant, being careful not to disturb the pellet (very gentle suction is recommended). The pellet can then be used for the assay. The amount of the protein solution and volumes of reagents can be lowered if there is too much protein in the sample for the assay, or if the volume of sample available is insufficient.
Thank you, Adam. This is quite useful, and I will try to precipitate the protein followed by the Bradford or BCA assay.
Thank you, Katja. My protein of interest is a silk protein. Individually, it can be estimated with the help of BCA / Bradford assay. But in composite form, false results were coming. I will try to read at 280 nm also.
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