I isolated mitochondria using Sigma-Alrich Mitochondria Isolation kit for Cultured cells (MITOISO2) and resuspended the mitochondria pellet using their Storage Buffer (50 mM HEPES, pH 7.5, containing 1.25 M sucrose, 5 mM ATP, 0.4 mM ADP, 25 mM sodium succinate, 10 mM K2HPO4, and 5 mM DTT). To quantify the mitochondrial protein content, I conducted BCA assay (Pierce). For BCA assay, I diluted my mitochondria by 5-fold with 2 different buffers - Storage buffer and DPBS. The results I obtained were quite different - 1.28 mg/ml for DPBS and 5.95 mg/ml for storage buffer. I think the observed discrepancy is due to the presence of DTT (a strong reducing agent) in the storage buffer. However, according to thermofisher's Protein quantitation assay compatibility table, it states that maximum compatible concentration for DTT that can be used in BCA assay is 1 mM. The storage buffer used was diluted to ~0.8 mM which means that it should not be affecting my BCA results. Therefore, I am wondering what is affecting the results i am getting from the BCA assay.
Hello Xiao,
it may be possible, that the sucrose concentration in the storage buffer is too high. I found a table with compatible concentrations and based on this table 1.25 M sucrose could be too high.
We have used a different mito-isolation kit (Qproteome® Mitochondria
Isolation from Qiagen) and also found that the storage buffer is not compatible with BCA.
Good luck!
Gisela Beutner Hello Professor,
I use the Thermofisher Kit for Isolation of Mitochondria and they haven't provided any storage buffer with the kit. So I was wondering what buffer I could use to resuspend the pellet in before BCA analysis. The manufacturer's protocol mentions 2%CHAPS solution for BCA but I was looking for alternative solutions.
The discrepancy in protein content measurements between the two buffers may be due to a few factors. The presence of DTT in the storage buffer at a concentration of 5 mM may be affecting the BCA assay results, as DTT can interfere with the colorimetric measurement of protein. Additionally, the presence of sucrose in the storage buffer at a concentration of 1.25 M may also be affecting the BCA assay results, as high concentrations of sugars can also interfere with protein quantification assays.
Another possible explanation for the discrepancy in results could be the difference in pH between the two buffers. The storage buffer has a pH of 7.5, while DPBS has a pH of 7.4. Although the difference in pH is small, it could still potentially affect the BCA assay results.
It is also possible that the mitochondrial isolation procedure itself could have affected the protein content measurements. The mitochondrial isolation procedure can lead to damage or loss of mitochondrial proteins, which would result in a lower protein measurement.
In conclusion, the discrepancy in the results could be due to a combination of factors including the presence of DTT, sucrose, and pH difference between the two buffers, or damage to the mitochondrial proteins during isolation. To confirm the reason for the discrepancy it is recommended to repeat the BCA assay in the absence of DTT and sucrose and using a buffer with a neutral pH. Additionally, you can test the mitochondrial integrity by using mitochondrial specific enzymes like NADH dehydrogenase or citrate synthase.
There are several factors that could potentially affect the results you obtained from the BCA assay:
To address these issues, you can try diluting your samples to different concentrations and testing them with the BCA assay, using a range of buffers and pH values. You can also consider using an alternative protein quantification assay, such as the Bradford assay or the Lowry assay, to cross-validate your results. Additionally, make sure to follow the manufacturer's instructions carefully and use high-quality reagents and equipment to ensure accurate and reproducible results.
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