Data analysis

What is Data analysis?

The data generated from a Western Blotting is quite straightforward. Usually, after the blot is treated with a suitable substrate, the bands of the target protein become visible and their origin can easily be confirmed by simply putting them side to side with a molecular weight marker or a positive control. But not all experiments are perfect. Sometimes, multiple bands, alterations in the bands and unusual sizes can make the data more complex.

Quantitation:

To begin with, it’s really important to understand that Western Blotting is a semi-quantitative process. It is not an absolute measure of the quantity of a given protein.
The main reason for this is that human error is present in loading and transfer rates that need to be normalised prior to precise comparison and that sometimes the signal is not linear across the concentration range.

Normalisation:

If the experiments is performed in order to compare the expression levels of the desired protein across more than one sample on the same blot or on different ones, using a loading control to normalise the data is a must. This way the differences in gel loading and transfer rates can be normalised. You can carry out an experiment without them, but in the case of publishing work or comparing the signal level is required, using them helps greatly. Most of the time the controls measure the levels of actin, histones, tubulin or GAPDH. Keep in mind the differences in molecular weight when choosing a loading control for a specific experiment, because a signal variation might occur.

Software and densitometers:

When a permanent record of the blot is needed, a densitometer can be used to scan or film the blot. After this process, imaging software is used, so the person can compare the generated band signal from the blot.

How to detect and characterize a protein?

The simplest use of a Western Blot is to determine the presence or absence of a target protein in a sample. This type of test is performed mainly to check for an endogenous expression of the protein of interest or to confirm if a transferred cell line is successful. Furthermore, it can be combined with protein purification and cellular fractionation so choosing which samples are good and which ones you should discard becomes an easier process. A straightforward way to check if the band corresponds to the desired protein reacting to the corresponding antibody is via using a competition peptide matching the epitope’s sequence. This experiment is performed during the incubation process, and an antibody is put side to side with the same one, but treated with a blocking peptide. The downside of this is that it can be used only for monoclonal antibodies, because they are single epitope directed.

The Prion disease:

It is a very rare neurodegenerative disease that is observed in humans, cows, sheep and deer. It is described with a fast loss of neurological functions and very characteristic brain lesions. These symptoms are seen after a long latency period. Several ways of identifying the prion protein responsible for this disease exist. Bioassays, immunohistochemistry and a targeted Western blot are some options.

HIV detection:

HIV detection: When a suspected HIV patient is first tested, an ELISA kit is used for the initial diagnosis, mainly because of its high specificity and ease of performing a substantial number of samples. On the flip side, this kit is prone to false positives, so a Western Blot is used as a second verification. This experiment is carried out by using the serum of the patient as the source of antibodies and HIV containing blots, peptides and proteins can be used to derive the target protein. If the proteins are detected by the serum, it can be said that the patient has a true HIV infection because his body reacted in the test. This process has proved itself to be useful not only for this infection, but also for Lyme and other autoimmune diseases.