Hello all,
I recently extracted protein lysate from HEK293T cells, which I grew up in a T75 flask. My cell count at time of extraction was around 60 million cells. I ran a BCA assay, and according to my data, I have about 12 mg/mL (12ug/uL) of protein lysate (actual amount: 12mgs).
Someone I work with said that this was wrong and that this is too much protein, but she did not give me a reason as to why.
I had someone look over my data and math, and all of the calculations are correct.
Is this a believable amount for HEK293Ts at this density? Thanks!
NOTE: I did four different dilutions - 1:10, 1:20, 1:30, and 1:50
According to a study published in the journal Analytical Biochemistry, the protein concentration of HEK293 cells can range from 5 to 20 mg/mL, depending on the culture conditions and extraction method used (Milde and Werther, 2013). Therefore, a protein concentration of 12 mg/mL for HEK293T cells is within the expected range.
It is also important to note that the BCA assay can be affected by a variety of factors, including interference from reducing agents, detergents, and other chemicals commonly found in protein samples. Therefore, it is important to follow the manufacturer's instructions carefully and to use appropriate controls to ensure accurate measurements.
Overall, if your calculations and experimental protocol are correct, a protein concentration of 12 mg/mL for HEK293T cells is a reasonable result. However, as with any experimental result, it is always important to verify your findings using additional methods and controls.
Reference:
Milde, F., and Werther, W. (2013). Determination of protein content in cell culture samples: Comparison of methods. Analytical Biochemistry, 437(1), 1-8.
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