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A Western blot with different types of background.

Do you sometimes wonder if you are taking a psychological test when you are trying to interpret your Western blot results? Black spots, smudges and speckles can make it difficult to produce publication quality images. Blotchy, uneven or speckled background can be due to a variety of things. For an in depth discussion of Western blot background causes and solutions, see our October Wiki topic. For a few quick trips to reduce uneven background, keep reading

Keep out the dirt

This might seem like a silly thing, but keeping things clean will go a long way towards getting rid of random background speckles. Enhanced chemiluninescent (ECL) reagents are attracted to dust and dirt. Wash all trays and equipment, keep covers on trays during incubations, filter buffers to get rid of particulates and be careful when moving the membrane around and don’t let it touch surfaces that might get the membrane dirty.

Get the air out

Air bubbles on the membrane can prevent proteins from sticking to the membrane, prevent diffusion of liquids and interfere with antigen:antibody interactions. When setting up your stack, make sure all air bubbles are pressed out by rolling a pipette over each layer. Be aware of other steps that generate bubbles: use older transfer buffer to decrease the generation of bubbles when methanol mixes with aqueous solutions, turn down the voltage during transfer, keep the buffer cool during transfer and don’t make bubbles by agitating solutions too much during incubations.

Be kind to your membrane

The membrane is a delicate support upon which your experiment is taking place. I have seen too many abused membranes in my years in the lab! Treat your membrane nicely and you will have nicer looking blots. Be careful not to bend, crease or scratch the membrane. Always use gloved hands to prevent transfer of skin oils. If using a polyvinylidene difluoride (PVDF) membrane, follow the manufacturer’s instructions to activate and equilibrate the membrane. Finally, never let the membrane dry out.

Get rid of ECL schmutz

If ECL reagents pool unevenly on the membrane they can give a splotchy, hazy background. A trick to prevent this is to lightly blot the membrane with filter paper before wrapping the blot in saran wrap; be careful not to let the membrane dry though.

We know you have wonderful Western blot results that you want to share. Follow these and other tips to make sure your blots are looking their best.

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