I do a 2-dimensional electrophoresis for a human tissue. My steps were as follows:
- Save samples in -20 Frizzier for 3 days.
- At the beginning of the SDS PAGE, we had to stop the running several times because of leakage from the upper buffer to the down buffer. Finally, blocking all seams, we successfully ran the SDS PAGE for about 1.5 hours after sitting IPGs on 12% gels.
- Problem: Unfortunately, after overnight staining by CBB R250, I found no protein spot; however, some smears appeared in the gels. I want to know why my gels did not have protein spots?
- *My finger hints to ladder place
The lack of visible protein spots in your SDS-PAGE after isoelectric focusing (IEF) could result from multiple issues, with potential causes including protein degradation, inadequate transfer from the IEF strip to the SDS-PAGE gel, or errors during staining. Specifically, the observed smearing could be due to protein degradation caused by prolonged storage at -20°C without sufficient protease inhibitors. Additionally, improper rehydration or focusing conditions during IEF might lead to uneven protein distribution or loss of proteins. Leakage issues during SDS-PAGE may have affected the separation, as interruptions in the electrophoresis process can disrupt protein migration. Lastly, staining inconsistencies, such as insufficient destaining or overstaining, might obscure protein spots. To address these issues, ensure the use of fresh protease inhibitors, optimize IEF conditions (e.g., protein concentration, rehydration duration), and verify the integrity of the gel assembly before running SDS-PAGE (Görg et al., 2004).
Reference: Article Current two-dimensional electrophoresis technology for proteomics
Thank you Debajit Das. Today, I performed an SDS-PAGE on my protein sample solved in the rehydration buffer. During the SDS-PAGE, I found that the samples moved backward into the upper buffer instead of into the gel. The electrodes were connected correctly. I do not have any idea about this phenomenon.
This phenomenon typically occurs due to a discrepancy in the pH or ionic composition of the sample buffer compared to the running buffer. If the rehydration buffer used for solubilizing your protein sample has a significantly different pH or ionic strength, it can alter the charge on the proteins, causing them to migrate in the wrong direction. To resolve this issue, ensure that your sample buffer is compatible with the SDS-PAGE running buffer. Additionally, verify that the buffer systems used are fresh and prepared correctly, as degradation or contamination can lead to abnormal migration. Another possibility is an issue with the gel itself, such as improper polymerization or incorrect preparation of the stacking and resolving gel. Reviewing these aspects should help prevent this anomaly in future experiments.
Thank you Debajit Das, your words are helpful and I am going to check the parameters related to running buffer pH and ionic strength of the rehydration buffer.