Protease Q: Process human synovial fluid samples for protease analysis and it turns to jelly. Think DNA is the cause, any suggesitons on how to fix?

If you are processing human synovial fluid samples for protease analysis and the samples are turning into jelly, it is likely that the cause is the presence of high levels of DNA. This can happen when the samples are not properly processed or stored before analysis, and the DNA is not removed or degraded.

Here are a few suggestions on how to fix the issue:

Sample preparation: Make sure that you are using fresh samples that have been collected and processed properly. Avoid prolonged storage of samples at room temperature or at high temperatures.

DNA removal: Use a DNA-specific enzymatic treatment, such as DNase, to degrade the DNA in the sample prior to analysis. Alternatively, you can use a commercial kit specifically designed for DNA removal from synovial fluid samples.

DNA quantification: Before proceeding with the protease analysis, you can quantify the DNA in the samples by using a fluorometer, or a spectrophotometer. This will allow you to estimate the amount of DNA in the samples and evaluate the effectiveness of the DNA removal step.

Protein precipitation: You can use a protein precipitation method such as trichloroacetic acid (TCA) or acetone, to remove the remaining DNA.

Gel electrophoresis: Run the sample on a gel electrophoresis to check for the presence of DNA.

Consider using a different technique: Alternatively, you may want to consider using a different technique such as mass spectrometry (MS) to analyze the proteases in the synovial fluid samples

Nikolay Klyashtornyy

If you suspect that DNA is causing your synovial fluid samples to become jelly-like, there are a few steps you can take to address this issue:

DNAse treatment: One possible solution is to include a DNAse treatment step before or during the processing of your synovial fluid samples. DNAse enzymes can degrade the DNA present in the samples and reduce its viscosity. You can follow the manufacturer's instructions for DNAse treatment and optimize the concentration and duration of the treatment to achieve the desired results.

Dilution: Try diluting your synovial fluid samples with an appropriate buffer or solution before centrifugation and heat denaturation. Dilution can help reduce the viscosity of the samples by lowering the concentration of DNA and other macromolecules that may contribute to the jelly-like consistency. You may need to optimize the dilution factor to find the right balance between reducing viscosity and maintaining the desired protein concentration for your protease analysis.

Extended centrifugation: If DNA or other macromolecules are causing the jelly-like consistency, you can try increasing the centrifugation time or speed to further separate the components. Experiment with longer centrifugation times or higher centrifugal forces to pellet any aggregated DNA or other materials that could be contributing to the jelly-like texture.

Filtration: After the initial centrifugation step, you can consider filtering the synovial fluid samples through a filter with an appropriate pore size. This can help remove larger DNA fragments or other particulate matter that may be causing the sample to become viscous.

Buffer optimization: Evaluate the buffer composition used during sample processing. Adjusting the pH, salt concentration, or the addition of detergents may help to prevent DNA from causing the jelly-like consistency.

It's worth noting that the viscosity of synovial fluid can vary between individuals and under different pathological conditions. Therefore, it is important to compare your samples with appropriate controls and consider the normal range of viscosity for synovial fluid during interpretation.

By implementing these strategies, you should be able to reduce the viscosity caused by DNA and improve the process of analyzing proteases in synovial fluid samples.

These video playlists might be helpful to you:

https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU

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