Western blot troubleshooting

Air bubbles

Caused by not placing the gel and membrane on the stack carefully enough or inadequate removal of air bubbles with a roller or plastic card. The air bubble creates a pocket where there is no buffer and therefore no conductivity. The sample will not be transferred from the gel to the membrane in this area and the membrane will look like it has small, sharply defined blank patches when stained with Ponceau S dye.

Poor conductivity

Since the electricity is conducted by ions in the buffer, insufficient soaking can lead to areas of poor conductivity. These areas tend to be larger and less well-defined than those caused by air bubbles.

Overheating

The experiment may overheat if there is increased resistance due to very poor conductivity or a lack of cooling. The former is caused by insufficient soaking with buffer, the latter by not including the ice pack during the transfer. This may result in the membrane burning or the gel melting. In areas where the gel has melted, no protein transfer will occur and these will appear blank when the membrane is stained with Ponceau S.

Wrong direction of migration

Proteins migrate from the cathode to the anode. The gel therefore needs to face the anode, and the membrane the cathode. If the stack is assembled in the incorrect order or the cassette is inserted into the tank the wrong way around, proteins could transfer in the direction of the filter paper instead of in the direction of the membrane. When stained with Ponceau S, the membrane will appear blank.

Layers not aligned

If the layers are not aligned properly or are not held tightly enough together they may move during the experiment. This will result in less sharply defined bands on the membrane when it is stained with Ponceau S.

Wrong orientation

Proteins are loaded onto the SDS-PAGE gel in a certain order. If the gel is rotated or flipped during the transfer step, samples may get mixed up. To avoid this, the gel should not look symmetrical. This can be achieved by cutting a corner. It can also be helpful to mark the membrane with a ballpoint pen.