In the hemolysis assays after incubating the erythrocytes with my protein, centrifuged to remove the cells and measured the absorbance of the supernatant at 490 nm (according to the protocol) but, what is measured at that wavelength? I would appreciate your response as I want to know the basis of this step.
i think this link will help you.
http://www.cyprotex.com/toxicology/mechanistic-toxicity/hemolysis
Hi Cinthia
the absorbance picks of Haemoglobin are different for type and/or for status of Haemoglobin (Oxy- Carboxy-, deoxy-, sulphaemoglobin) as well as the pH of the medium. Even the binding with protein can change the spectrophotometric properties of HB. My be the suggested wavelength for your test consider the different variable in the final hemolysed sample (added protein, final pH, etc)
Dear Cynthia, take a extra care when measuring the absorbance. Several articles says different absorbances. I think 490nm is the peak for the haemoglobin. But if you spectrofotometer can do read several absorbances (a screening), put 250 to 600 and use the peak as the correct for you. Because, as Antonio said, several things can influence the absorbance
Dear Cynthia,
That wavelength corresponds to the maximal absorption peak in the visible region. Please, see the following link
https://www.google.co.ve/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&ved=0ahUKEwjBjvW3s_zUAhXIE5oKHccKBf8QjRwIBw&url=https%3A%2F%2Fen.wikipedia.org%2Fwiki%2FPhotoacoustic_imaging&psig=AFQjCNHRcING2JkfO5N1m856p9M1gCW-rw&ust=1499696611047010
Regards,
Pedro.
Cynthia Martinez:
The least compromising wavelength to read hemoglobin OD in your sample is at 527 nm, which always is the isosbestic point for both oxy and meth hemoglobins, the most common species in your samples.
Peter K Lauf Sir what about 540nm for hemolytic assay ? as it is widely available and astm method for hemolysis assessment is also based on it.
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