Bands are lower or higher than expected in western blot

The gel has been run for too long.

Before proceeding with blocking and immunostaining, check the transfer of proteins to the membrane with Coomassie blue. If all bands appear very low, you may have left the proteins too long to migrate through the gel.

Run the gel according to the manufacturer's instructions.

• Ideal running times and voltages can vary according to the manufacturer, gel composition, and size of the protein of interest
• Running the gel at 100-150V for 1-2 hours in pre-chilled electrophoresis buffer is a general recommendation
• Frequently monitor the separation of ladder markers to adequately resolve within the estimated size and range of the protein of interest

Not enough acrylamide in the gel.

The bands may be very low on the blot if there's not enough acrylamide in the buffer. This is because the proteins do not experience enough resistance, so migrate too quickly across the gel.

• You should generally run lower molecular weight proteins in gels with a higher percentage of acrylamide.

• Check our western blot guide for suggested gel recipes, and increase the amount of acrylamide if necessary.

The gel has not been run for long enough.

If all bands appear very high, the proteins may not have had enough time to migrate across the gel. You can check this with Coomassie blue before proceeding with blocking and immunostaining.

• Try running the gel for longer before proceeding; see notes in above section.

Too much acrylamide in the gel.

The bands may be very high on the blot if there's too much acrylamide in the buffer, as high acrylamide density can hinder effective migration of proteins through the gel. You should generally run higher molecular weight proteins with a lower percentage of acrylamide.

• Check our western blot guide for suggested gel recipes, and reduce the amount of acrylamide if necessary.