Hello Isaiah Burciaga

You should get around 5-10mg/ml protein from liver homogenate.

FYI: A mouse liver weighs around 2-3 grams. It yields approximately 2-4 mg of protein per gram of tissue. So, the total protein yield would be around 4-12 mg of protein from the entire liver.

Getting 50mg/mL is indeed a large amount.

How was the condition of the mouse?

Usually, fatty liver disease can disrupt the normal flow of proteins and fats within the liver, contributing to higher protein concentrations. A high-protein diet can also lead to an increased influx of amino acids into the liver. If there is a deficiency of certain liver enzyme, it is likely to lead to metabolic imbalance.

Also, there could be other reasons for such a large amount of protein. Check for the following

1. Check for contamination as the liver homogenate might have been contaminated with other proteins, causing an artificially high reading.

2. Revisit your protocol as there are some protocols that include steps to concentrate the protein.

3. The protein assay itself could have a problem (e.g., a reagent error or something similar), it could lead to misinterpretation of results.

Best,

Hello, Isaiah Burciaga.

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.

See more details:

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

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 higher 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. See the MCE product webpage for the limit concentration.

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 in advance and restore it to room temperature before use to improve the sensitivity of the detection.

If you have any other questions, please contact me.