Not very sensitive, lots of protein required. Advantageįast, cheap, visual, no specialist equipment required This is far more protein than is necessary in other methods, which can detect a signal in the femtogram range. The main limitation of colorimetric detection lies in its s ensitivity, with protein in the nanogram range required to produce a signal. By contrast, AP in colorimetric detection produces a stable substrate that will not fade and allows the use of different substrates to produce different colors on the same membrane. HRP in colorimetric detection is v ery cost-effective but may fade on exposure to light and can produce n on-specific staining. Stop development by washing the membrane, judged by your own approximation.Insoluble precipitate accumulates on and stains the membrane, visible to the eye.Add appropriate substrate to membrane probed with antibodies.In colorimetric detections, the enzyme conjugated to the secondary antibody triggers a reaction with a substrate to produce a colored precipitate, according to the following steps: A proper loading control must be used in the experiment if you want to be able to conduct any quantitative analysis using densitometry methods. The images produced can be analyzed quantitatively by densitometry (intensity of signal) or by spectrophotometry. The secondary antibodies are conjugated to either horseradish peroxidase (HRP) or a lkaline phosphatase (AP) and can be used in either colorimetric or chemiluminescent detection methods. This method uses secondary antibodies that are conjugated to enzymes and therefore require a substrate to work. Health and safety risks, expensive, time-consumingĮnzymatic detection Colorimetric and Chemiluminescent The film develops a dark band with exposure to the radioactive tag relative to the weight and amount of protein present.A band of protein is detected by exposing an X-ray film to the membrane, often for up to 48 hours.This radioactive detection method began declining in popularity as other methods were developed, owing to the hazards associated with exposing scientists to radiation. Historically, antibodies for Western blot were tagged with radioactive labels. Understanding the different options, how they work, and what the benefits are may make it that much easier to identify your protein and contribute to the success of your experiment. The method you choose will depend on your experiment and the availability of equipment in your lab. Over the years, the general technique has remained very similar, although the detection methods have been expanded and refined to allow for more precise and quantitative analysis of proteins.

On your membrane, these antibodies should produce a band specific to the molecular weight, which can be understood using the protein ladder run in conjunction with your samples in the gel. Proteins are separated by size through a gel by electrophoresis, transferred to a membrane and then identified using primary and secondary antibodies. The Western blot is a widely used technique that has been around for over 40 years.

Western blot technique