Troubleshooting Tips for Western Immunoblotting

The following tips can be used to overcome the most common problems encountered during Western blotting.

Smeared Pattern or Distorted Bands

• Uneven contact between gel and membrane: cassettes used should allow a tight fit, leading to even pressure over the entire surface of the gel and membrane.
• Gel not equilibrated in buffer prior to transfer: the gel should be soaked in transfer buffer containing methanol for 15 to 30 minutes, in transfer buffer without methanol for 60 minutes before assembling the transfer sandwich.

"Bald Spots"

• Bubbles between gel and membrane: bubbles create points of high resistance that lead to low transfer efficiency, therefore care should be taken to remove bubbles completely when putting together the transfer sandwich.

Incomplete Transfer

One of several technical errors can be the source of this problem:

• Proteins not completely eluted out of gel: this often occurs with high molecular weight proteins, especially when using a transfer buffer containing methanol. One way to overcome this phenomenon is by using a nylon membrane, which does not require methanol in the transfer buffer. Adding SDS to the transfer buffer as well as using higher field strengths also improves protein elution.
• Proteins have transferred through membrane: this may occur when working with proteins of very low molecular weight. Optimizing/shortening transfer times and using a double layer of membrane usually enables retention of small proteins.
• Inappropriate transfer buffer used: the most stable and commonly used buffers are Tris-Glycine based.
• Impurities in the transfer buffer: this will lead to a pattern on the membrane that mirrors the holes in the transfer cassette. Fresh buffer should be prepared prior to each transfer process.

High Background

• Cross-reactivity between blocking agent and primary or secondary antibody: this will result in an overall membrane staining. Usually, the addition of a mild detergent such as Tween-20 to the incubation and washing buffers will eliminate the problem. If background staining persists, changing the blocking agent is recommended.
• Concentration of antibody too high or incubation time too long: the higher the antibody concentration and the longer the incubation time, the greater the likelihood for non-specific staining. Raising the incubation temperature (e.g. to 37¡ C) is recommended over lengthening the incubation time. Also, several short washing steps are better than one long one.
• Membrane dry during incubation process: care should be taken to keep membrane from drying out during incubation.

Little or No Signal

• Antigen is not recognized by primary antibody: this can occur especially with monoclonal antibodies that were raised against a native protein. In some cases, a non-reducing gel system may need to be used.
• Inhibition of secondary antibody conjugate: Horseradish peroxidase labeled antibodies should not be used in conjunction with sodium azide or hemoglobin. Biotinylated antibodies should not be used with milk or casein since both contain biotin. Concanavalin labeled antibodies should not be brought into contact with milk due to interfering glycoproteins. A change in the blocking agent or incubation solution will solve this problem.
• Detergent is too harsh: SDS, Nonidet P-40, and Triton X-100 disrupt binding between proteins. Tween-20 is the most commonly used and recommended detergent for washing and incubation solutions.