Your observation of ~50 mg/mL total protein from mouse liver lysates using RIPA buffer and the Bradford assay is notably higher than expected. Typically, protein concentrations from liver tissue range between 5–15 mg/mL under standard extraction conditions.Several factors may be contributing to this apparent overestimation: Assay interference: RIPA buffer contains detergents (e.g., SDS, Triton X-100) known to interfere with the Bradford assay, often leading to artificially elevated readings. Dilution errors: High dilution factors (e.g., 1:100) increase the likelihood of pipetting inaccuracies. Lysis conditions: A low buffer-to-tissue ratio may result in overly concentrated lysates. Sample clarity: Incomplete lysis or residual debris can skew absorbance readings.

Recommendations: Consider switching to a BCA assay, which is more compatible with RIPA. Prepare Bradford standards in the same buffer as your samples. Validate results using multiple dilution points. Ensure thorough centrifugation before quantification.

Overall, your results suggest an overestimation likely due to buffer interference or technical variability, both common issues with Bradford assays in RIPA-based extractions.

Sumia Mohamed Essid Thank you so much for your detailed response — that really helps clarify things!

It’s reassuring to know that 50 mg/mL is unusually high and that assay interference is a likely culprit. I suspected the RIPA components might be affecting the Bradford readings, especially since we're using a standard buffer with SDS and Triton X-100.

To answer some of your points:

• We did notice some variability between replicates, likely due to the high dilution factor, so your point about pipetting accuracy at 1:100 makes sense.
• We’ve been using RIPA buffer at a 1:10 (w/v) tissue-to-buffer ratio — maybe that's on the lower side and contributing to higher concentrations.
• I’ll definitely try preparing the Bradford standards in the same RIPA buffer next time to better match the sample matrix.
• And yes, we’ll look into switching to the BCA assay for future experiments — thanks for that suggestion.

I’ll also make sure to centrifuge thoroughly before reading the absorbance, as we might have some residual debris affecting the results. Thank you for you help again!

Protein concentrations as high as 50 mg/mL from mouse liver lysates seem unusually elevated and may indicate interference with the Bradford assay, especially if using RIPA buffer containing detergents like SDS or Triton X-100. These components can artificially increase absorbance, leading to overestimation. Typically, total protein concentrations from liver tissue fall in the range of 1–10 mg/mL after extraction. I recommend preparing a standard curve using the same buffer (RIPA + inhibitors) and validating results with an alternative method like the BCA assay, which is more detergent-tolerant. Also, serial dilutions can help detect nonlinear responses due to buffer effects.

Fan Janice ,

It is probably abnormal. According to the normal extraction process and the appropriate tissue to buffer ratio, the protein concentration in the extract is usually around 1-10 mg/mL. Generally speaking, 20 mg of frozen mouse liver tissue may produce 15-25 mg/mL protein.

Reference:

(Protocols from MCE) https://www.medchemexpress.com/inhibitor-kit/ripa-lysis-buffer-(strong).html

I have some small suggestions for extraction, I don’t know if it helps you.

1. Check the ratio of tissue volume to RIPA buffer:

If too much liver tissue is added during extraction and too little RIPA is added, the protein concentration in the extract will be too high. Because the insufficient volume of lysis buffer will cause a concentration effect.

In particular, the RIPA buffer (Cat.NO.: HY-K1001) provided by MCE recommends adding 150-250 μL RIPA to 20 mg of tissue. Our product is cost-effective and lyses quickly. If the experimental object is mouse liver (about 1.5 g), 11.25 mL-18.75 mL RIPA buffer needs to be added.

2. Is the tissue homogenization insufficient:

Judge whether the solution is clear and whether there are impurities visible to the naked eye. Insufficient homogenization may cause the protein to not be completely released into the buffer. In subsequent operations, more protein will be gradually released over time or with oscillation, resulting in a high protein concentration when measured.

3. Is there any impurity interference:

During the extraction process, if impurities such as cell debris and lipids are mixed in, it may affect the results of the Bradford method and result in an excessively high protein concentration. For example, if cell debris or unlysed substances are not removed, particulate matter increases light scattering, resulting in an artificially high absorbance value.

4. Bradford compatibility issues with detergents and reducing agents:

The Bradford kit (Cat.NO.: HY-K2001) provided by MCE is compatible with a series of detergents and reducing agents, but too high a concentration will affect the test results. For the limit concentration, see the MCE product webpage.

Reference:

(Manual from MCE) https://www.medchemexpress.cn/inhibitor-kit/bradford-protein-assay-kit.html

5. Others

Before use, the standard protein solution must be completely dissolved, mixed and then diluted into a series of gradient concentration protein standard solutions. It is also recommended to take out the G250 staining solution and restore it to room temperature before use to improve the sensitivity of the detection.

The answer to this question comes from MedChemExpress (MCE) Technical Support.

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