Why Do Proteins Fail to Form Bands in Two-Dimensional Electrophoresis
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Low protein concentration: Ensure the protein concentration is sufficiently high, typically within the range of 5-10 µg/µl.
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Protein degradation: Prevent degradation by adding protease inhibitors and keeping the sample at low temperatures.
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Impurities in the sample: Excessive salts, lipids, or other contaminants can interfere with separation efficiency. Purify the sample or perform dialysis to remove unwanted substances.
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Inappropriate pH gradient: The selected pH gradient should encompass the isoelectric points of all proteins in the sample.
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Insufficient focusing time: Extend the isoelectric focusing duration to achieve optimal protein separation.
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Buffer-related problems: Verify that the IEF buffer has the correct pH and concentration, and always use fresh buffer solutions.
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Gel preparation errors: Ensure the gel is properly prepared with the correct concentration, avoiding bubbles and unpolymerized regions.
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Suboptimal electrophoresis conditions: Adjust voltage and running time to prevent excessive voltage from causing overly rapid protein migration or insufficient voltage from leading to incomplete separation.
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Instrument malfunction: Regularly check the electrophoresis system, including electrodes and the electrophoresis tank, to ensure proper functioning.
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Buffer issues: Ensure the buffer solution has the correct pH and concentration, and always use freshly prepared buffers.
Proteins may fail to appear as bands in two-dimensional electrophoresis due to several factors. Below are common causes along with possible solutions:
1. Sample Preparation Issues
2. Isoelectric Focusing (IEF) Issues
3. SDS-PAGE Step Issues
4. Electrophoresis Equipment Issues
5. Staining Issues
After electrophoresis, proper staining is necessary to visualize protein bands. If the staining reagent is of poor quality or the staining duration is insufficient, the protein bands may not become clearly visible.
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