After protein cross-linking, cell lysis was performed using the lysis buffer in the Biyuntian reagent kit, but the concentration measured by the BCA method is very low. What could be the reason?
1. When determining protein concentration using the BCA method after protein cross-linking, if the resulting concentration is very low, there may be several reasons:
1. Cross-linking effect: Protein cross-linking may cause changes in the structure of the protein, which can result in certain detection sites becoming blocked or unable to effectively react with the reagent, leading to a concentration value lower than the actual one.
2. Reagent reaction conditions: The BCA method is very sensitive to reaction conditions, including pH, temperature, and reaction time. Make sure to strictly follow the instructions provided with the kit, ensuring that all conditions are appropriate.
3. Influence of lysis buffer: The lysis buffer used may affect the sensitivity of the BCA method. For instance, if it contains certain chemical components (like reducing agents or some protease inhibitors), it might interfere with the BCA reaction.
4. Sample dilution: If the sample is overly diluted before measurement, it may result in the final concentration being below the detection range, thereby affecting the results.
5. Expired or improperly stored kit: Ensure that the kit is within its expiration date and stored under appropriate conditions, as improper storage can affect experimental outcomes.
2. You can try the following methods to further confirm the problem:
1. Attempt using other protein concentration determination methods, such as the Bradford method or Lowry method, for comparison.
2. Change the composition of the lysis buffer to see if there is any improvement.
3. Review the experimental steps to ensure all operations are conducted under recommended conditions.
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