I am trying to purify a stroma-free lysed RBCs for hemoglobin isolation using 14 KDa dialysis bags. Using BCA assay for total protein quantification before the SDS-PAGE, I got the following concentrations:
Stroma-free lysed RBCs: 13.1 +/- 0.4 mg/ml.
Hemolysate: 16.3 +/- 0.2 mg/ml.
Do you have any explanation for these results?
The increase could be false increase. It could as a result of interference from other released substances in the haemolysate.
Great thanks for your suggestions. But hemoglobin is a protein, so it will normally react with BCA reagent.
Do you mean that the unreacted hemoglobin will show absorbance at the same wavelength?
Great thanks, Dr. Harald
When I omitted the measured absorbance due to Hb on the UV-Vis spectrogram, the calculated concentration due to the BCA assay reached 15.138 mg/ml.
Is this result logical with respect to the dialysis product?
Thanks Dr. Harald for your support. Quantification of the total proteins using the nanodroper is a very good idea.
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