I extracted proteins from mouse thyroid tumors with RIPA buffer. Some lysates are red and I would like to remove hemoglobin befor protein quantification (BCA assay). How can I do it?
You could use an anti-hemoglobin antibody to immunoprecipitate the hemoglobin. Ideally, the antibody would be attached to a solid support such as beads, which could be spun down to remove both the hemoglobin and the antibody.
Thank you very much for your suggestion. I was thinking about a possible solution capables to degradate the hemoglobin. Do you know something like that?
Thank you again
Hello Erica, I know that this is not the answer to your question, but maybe next time you can use any RBC Lysis buffer (we use the Biolegend one) to get rid of erythrocytes before the protein extraction. We use it in the dispersion of mouse/human tumours
We use it in the dispersion of mouse/human tumours after the mechanical/enzymatical disruption, so we use it in a cell suspension. I don't know if it works with bigger pieces of tissue.
Good luck with your experiments!
Hi Alejandro,
hope you are doing well! Thank you very much for the answer, it is very helpful!
Degrading the hemoglobin would require adding proteolytic enzymes. These would also degrade the other proteins in the lysate, but would not remove the hemoglobin-derived peptides, which would still contribute to the total protein concentration. I'm not sure what would happen to the heme. Maybe it would precipitate once the hemoglobin was thoroughly degraded, maybe not. This doesn't sound like a good solution to me.
In any case, the red color itself may not be a problem for the BCA assay, as long as it is not too much. The assay is quite sensitive, allowing protein quantitation as low as 1 µg, so the amount of your lysate needed for the assay may be so small that the red color is of no consequence. The usual wavelength of the BCA assay readout is 562 nm, and the extinction coefficient of oxyhemoglobin at 562 nm is 0.033 per µM per cm (source: http://omlc.org/spectra/hemoglobin/summary.html). However, you could read the assay at 600 nm instead, reducing its sensitivity by a factor of about 2, but reducing the hemoglobin absorbance by factor of 10 (extinction coefficient is 0.003 per µM per cm).
Dear, lasma hemoglobin is depleted using Biotech Support Group's HemogloBind™ to extract hemoglobin from lysed red blood cells. Hemoglobin removal was accomplished by using HemoVoidTM depletion reagent.
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