Question 1

How to Analyze Western Blot Data ?

Accepted Answer

The data produced with a Western blot is usually quite easy to interpret. In the majority of cases, bands corresponding to the target protein will become visible upon treatment of the blot with substrate. Their identity is confirmed by comparison to molecular weight markers (for size) and a positive control (size and signal). In some cases the data may be more complex, showing unexpected sizes, multiple bands, or alteration in bands following a particular treatment. To estimate the molecular weight of the protein you can make a comparison with marker proteins and the amount of protein can be determined as this is related to band intensity (within the limits of the detection system).  In most applications, it is enough to confirm protein presence and roughly estimate the amount. However, other applications demand a quantitative analysis that defines protein levels in either relative or absolute terms.

Question 2

How Would You Describe Western Blot Data?

Accepted Answer

To know how to analyze western blot data, Look for the sizes of the bands. These will be represented by a number, either followed by "kDa" or preceded by "p." This is the size of the protein which has been detected and is the scale on which the proteins are separated in a Western blot. It is these differently numbered bands that represent different proteins and will determine a positive result or not. Determine which bands have a positive result and what this may mean. For example, with the test for Lyme disease, there are several different bands which may produce a positive result, and any one of these will mean a positive result. Other tests may simply produce non-significant bands. These may not be reported, but ensure it is clear which are the important bands. Specific bands which show a positive result means a positive result for the infection that was being tested for. Discuss the results and any concerns or questions with a clinician. A positive result may require some treatment, and the consequences of this should also be discussed with the clinician who ordered the Western blot diagnostic test.

Question 3

Can Western Blot Be Quantitative?

Accepted Answer

Western blot is a reliable quantitative method only if sample properties and integrity, antibody specificity to the target protein, and loading protocols are considered. With careful attention to details, you can avoid common mistakes and avoid misinterpreting Western blot data.