Table 1
From the following article
Southwestern blotting in investigating transcriptional regulation
Francis K Y Siu, Leo T O Lee & Billy K C Chow
Nature Protocols 3, 51 - 58 (2008) Published online: 20 December 2007
doi:10.1038/nprot.2007.492
Table 1. Troubleshooting table
| Problem | Possible reason | Solution |
|---|---|---|
| No bands observed after experiment | Poor quality of nuclear extract | Check the protein concentration
Confirm the DNA–protein interaction by performing electrophoretic mobility shift assay |
| Low protein concentration in the extract | Make sure protease inhibitors are added in Steps 3 and 6
Use more cells for nuclear extract isolation Reduce the volume of nuclear extraction buffer in Step 6 Prepare nuclear extract by other methods14 | |
| Inefficient labeling or weak radioactivity | Check the labeling efficiency and prepare a new probe
Use fresh 32P within the first half-life | |
| Protein not transferred to the membrane | Check electroblotting set-up
Extend the transfer time if necessary | |
| Probe is too short | Use a longer probe, but note that too long a probe may cause nonspecific binding | |
| Probe concentration is too dilute | Reduce the volume of TNED buffer in hybridization
Increase the amount of probe in the hybridization | |
| The expression level of the target protein is too low | Increase the amount of nuclear extractUse a longer exposure time and use intensifying screen | |
| Too many bands are observed | Nonspecific interaction | Reduce amount of extract and/or probe |
| Probe is too long | Redesign the probe
Use shorter oligos, but not <18 bp | |
| Bands compacted on top of the blot | Size(s) of the target factor(s) is/are large | Use a longer running time in SDS-PAGE
Lower the percentage of separating gel in SDS-PAGE |
| High background | Contamination of membrane | Handle the membrane carefully
Wear gloves and clean the containers/chamber before use Filter all solutions |
| Bubbles trapped during electroblotting | Remove all bubbles trapped in the electroblotting setup | |
| Evaporation of the hybridization buffer | Seal the hybridization chamber/box tightly | |
| TNED buffer with 5% skim milk turned yellow or some precipitate observed | Prepare fresh solution
Make sure incubation in light-tight condition | |
| The molecular weight of the band is difficult to determine | Too long or too short running time | Adjust run time |
| Poor resolution of the separating gel | Use higher or lower percentage of gel | |
| The color of the protein ladder faded out | Mark the ladder on the blot using pencil after electroblotting |