Hi all!
I have to quantifiy some proteins in RBCs by ELISA. Since RBC number cannot be standardized and it obviously affect the results, there is the need of some normalization. I read that many groups normalize for total protein content measured by CBA assay. However, I was wondering whether I can normalize my values by using haemoglobin, which accounts for 98% of total proteins in RBCs. For me, it would be far less time and sample consuming, since I would use a spectophotometer and very few microliters of sample, but I would like to know if someone ever used this normalization and if can be considered as correct and reliable.
Many thanks in advice!
Using hemoglobin for normalization in red blood cells (RBCs) instead of total protein content is a common practice in certain types of experiments or analyses, particularly those focused on hematology or erythrocyte physiology. Here are some considerations for using hemoglobin as a normalization factor:
Overall, using hemoglobin for normalization in RBCs can be a valid and informative approach, especially in studies focusing on erythrocyte physiology or hematology-related parameters. However, careful consideration of experimental design, validation of measurement methods, and comparison with alternative normalization factors are essential for ensuring accurate and meaningful results.
Dear Kais Khudhair al Hadrawi
thanks for your kind and thorough reply.
In RBC, I am actually looking for proteins to be used as biomarkers for diseasese not related to RBCs. Therefore, RBCs for me are just a biological matrix, and the proteins I am going to investigate should be not related to RBC function. I can consider my sample as sufficiently pure, since I careflly wash it many times to remove serum and carefully remove the white ring of leukocytes. The reason why I would like to normalize for haemoglobin content instead of total protein is because, as you mentioned, it is simpler, faster, more standardized and requires far less amount of sample.
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