Can the quality of protein transfer be checked before the blot is developed with antibodies?

Save time and check gel electrophoresis and protein transfer quality with this simple method:

Following Western blot, stain the membrane with reversible stains, like: Coomassie R250 or Ponceau S.

Destain in water and visually assess the quality of electrophoretic protein separation and transfer.

If the bands are fuzzy, distorted or smearing (as in the example to the right), review used protocol in respect to the following conditions:

Sample preparation

Incomplete protein denaturation due to lack of reducing agents such as DTT or beta mercaptoethanol.
Too high temperature used for sample denaturation, which leads to formation of aggregates, which are stacked at the top of the gel.
Overloading of protein amount/well can lead to smeared and fuzzy bands.

Gel electrophoresis and protein transfer:

Too high voltage during gel electrophoresis. Uneven protein migration and blurry bands.
Air bubbles, trapped between gel and a membrane, create blank and fuzzy areas.

Blocking and washing
Insufficient blocking and washing steps contribute to non-specific antibody binding

Easy check of protein transfer in Western blot
Quality of gel electrophoresis and protein transfer in the blot to the left is appropriate, and therefore this blot will be used for blocking and antibody incubation.

Quality of the blot to the right is questionable and continuing with this blot will be a waste of time.

Short video

Webinars on demand, explaining Western blot technique with specific examples of what can go wrong and how to solve the problem.

Can the quality of protein transfer be checked before the blot is developed with antibodies?

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