Greeting alls! I would like to ask on how can I determine the protein concentration of my purified outer membrane proteins (OMPs) if the results shows through the spectrophotometer are in negative values.
Negative values indicate a methodological problem, or some interference in the assay by some component of the sample (e.g., detergent). A different assay method, such as the BCA assay may be needed if the sample interferes with the Bradford assay.
A negative absorbance value from a spectrophotometer is not physically meaningful and is likely due to experimental error, instrument malfunction, or incorrect settings. Here are some steps to troubleshoot and address this issue:
1. **Check for Errors:** Make sure you have followed the correct procedure for preparing and measuring your sample. Check that you are using the correct cuvette, setting the right wavelength (595 nm in your case), and calibrating the instrument properly.
2. **Sample Preparation:** Ensure that your sample preparation was accurate. Verify that you used the correct volume of the sample and reagents. Check for any contamination or improper mixing.
3. **Cuvette Contamination:** Ensure that the cuvette you're using is clean and free from scratches. Even small contaminants or defects can affect the measurement.
4. **Instrument Calibration:** Confirm that the spectrophotometer is properly calibrated. If it's not calibrated, it can lead to inaccurate readings.
5. **Wavelength Settings:** Make sure you have selected the correct wavelength for the measurement (595 nm). Using the wrong wavelength can result in incorrect readings.
6. **Quality of Reagents:** Check the quality and freshness of the reagents you're using. Expired or degraded reagents can lead to unexpected results.
7. **Light Path and Cuvette Filling:** Ensure that the cuvette is filled properly, and the light path is clear. Air bubbles or uneven filling can distort readings.
8. **Instrument Maintenance:** Regularly maintain and service your spectrophotometer according to the manufacturer's recommendations.
If you've gone through these steps and are still getting negative absorbance values, it's advisable to consult with experienced colleagues or the technical support of the instrument manufacturer for further guidance. Negative absorbance values are generally not physically meaningful, and resolving this issue is crucial to obtaining accurate protein concentration measurements.
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