Hello all,
I measured protein contents before and after hydrolysis, but the result is very weird......
The initial protein concentration was around 2 mg/ml, and the limited concentration for my BCA is below 1 mg/ml, so I diluted my samples in 1/2.5.
I'm wondering whether BCA method is also suitable for hydrolysates or not? Does anyone have some ideas?
Thank you in advance.
Yuhong
The BCA assay relies on the reduction of Cu2+ to Cu1+ by peptide bonds, followed by detection of Cu1+ by bicinchoninic acid. Hydrolysis reduces the number of intact peptide bonds, which decreases the amount of Cu2+ that is reduced. Mild hydrolysis of large proteins shouldn't affect the protein concentration result very much. However, prolonged/complete hydrolysis can significantly change your BCA assay readout, especially if your starting protein is small.
Dear Shih-Hon, thanks a lot. So as you said, hydrolysis results in less peptide bonds, so that it should contribute to a lower readout? But in my case, the absorbances of one sample varied in a wide range, overall the readout of hydrolysates was much higher than that of protein without hydrolysis. I don't understand....
How did you hydrolyze your samples? Because if you used a protein (eg.trypsin) it can happen that you get a higher reading...although it is quite weird since it should happen what Shih-Hon Li said....
Dear Alessandro,
The hydrolysis was processed via a immobilized trypsin column, normally there should be no or little leakage of trypsin...
From the info provided, some possibilities for your higher post-column BCA reading are:
1. You have unanticipated protein leakage off of the column - You can run a sham experiment and see how much extra protein you get from the column itself. Either flow your sample buffer over or you could add some control protein like albumin. See if the collected sample has an increased BCA reading.
2. Something is interfering with the BCA assay - Double check the Pierce user guide to see if all of your buffers, including the column storage buffer, are compatible. Detergents like SDS commonly elevate the BCA result. Trypsin columns are commonly stored with a low pH buffer or with an amino acid like arginine. These could both be potential sources of interference.
3. The trypsin column is concentrating the sample - Is your total protein actually increased or just the concentration? You might be getting a size exclusion chromatography effect from your column.
Dear Shih-Hon,
I will try, and update my result later, let's see. Thank you for your advise:)
Yuhong
In our experiments alkaline hydrolysis of BSA (4 M NaOH, 48 h, +37 deg. C) led to almost 2-fold (BCA assay) and 1,3-fold (Bradford assay) decrease of absorbance in comparison with non-treated protein (incubated with equal volume of water in the same conditions). Details can be found in Fig S8 in preprint https://chemrxiv.org/articles/preprint/Measuring_the_Concentration_of_Protein_Nanoparticles_Synthesized_by_Desolvation_Method_Comparison_of_Bradford_Assay_BCA_Assay_hydrolysis_UV_Spectroscopy_and_Gravimetric_Analysis/13285712/1?file=25606697
Decrease can be caused by destruction of amino acids and peptide bonds that bind Coomassie and reduce Cu ions.
One more experiment, now with trypsin hydrolysis. We diluted gelatin B (75 bloom) to 2,1, 0.5... mg/ml in a) 0.1xPBS (pH circa 7), b) 0.1xPBS+ 10 ug/ml trypsin, c) 0.1xPBS+ 5 ug/ml. Gelatin solutions were incubated at +37C for 60 min then analyzed with BCA assay (25 uL protein+200 uL BCA reagent, +37C, 30 min in 96 well plates). Results in attachment. n=3 (technical replicates), mean+sd. At gelatin concentrations 1 and 2 mg/ml difference of absorbance values was 7% and less (p
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