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Bands appear very low or very high in Western blot

Explore possible causes and solutions for bands appearing very high or very low in Western blot.

Bands very low

Possible causes

Solution

The gel has been run for too long.

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

  • Try running a gel again for a shorter time before proceeding.

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.

Bands very high

Possible causes

Solution

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 Ponceau S before proceeding with blocking and immunostaining.

  • Try running the gel for longer before proceeding.

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 block 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.