Western Blotting Data Analysis | Bio-Rad (2024)

FAQs

How do you statistically analyze Western blot data? ›

To this end, once a western blot has been imaged, it can then be analyzed. To do this, researchers use a technique called densitometry to measure the relative amount of a specific protein for a given experimental sample on the blot by comparing it with a control or another protein.

AzureSpot Pro combines several powerful analysis tools into one convenient and easy-to-use package. Designed to guide you through the analysis process, AzureSpot Pro is an easy-to-use Western blot image analysis software that makes complex and customized analysis simple.

When presenting a western blot in a Starr lab meeting or presentation, include the following information: Title: Date, protein(s) and cell lysates including conditions being analyzed. Subtitle: Your initials, and date where details can be found in your lab book (see Lab Book Details).

The other common approach for quantitating western blots is commonly referred to as using lane and band tools. This approach utilizes a two-step procedure where each lane of a blot is identified in the image, and then individual bands are identified inside each lane.

To perform a Western Blot successfully, every single step should not be neglected. It includes: (1) WB buffers preparation, (2) samples preparation, (3) gel electrophoresis, (4) protein transfer, (5) membrane blocking, (6) antibody incubation, (7) WB detection and imaging, (8) Data analysis.

Unlike the PCR assay, Western blot analysis provides direct evidence for the presence of specific proteins. However, similar to RT-PCR, care should be taken when preparing samples for Western blot analysis and immunohistochemical detection (described next).

Maximum blocking time should not exceed 2 hours at room temperature. Any longer and proteins can be exchanged from the membrane. Another important factor you should consider when Western blotting is the integrity of the digital imager you're using. It is important to use a system that provides clear results.

A qualitative Western blot shows the presence or absence of a protein of interest but provides no information on relative protein abundance. A quantitative Western blot is used to detect specific proteins and measure relative protein abundance.

The western blot is extensively used in biochemistry for the qualitative detection of single proteins and protein-modifications (such as post-translational modifications). At least 8–9% of all protein-related publications are estimated to apply western blots.

What imager is used in western blot? ›

Imagers used in Western blotting include gel documentation systems, which visualize acrylamide gels following protein separation. Imagers for chemiluminescent detection offer high sensitivity, signal stability, and ease of use.

Five steps are involved in western blotting procedure and detection assay, namely, transfer, blocking, primary antibody incubation, secondary antibody incubation and protein detection, and western blotting analysis.

In order to visualize proteins on the membrane the antibodies are typically conjugated to an enzyme, such as horseradish peroxidase (HRP), that emits light upon reacting with a specific substrate. The emitted light, or chemiluminescence, is detected on x-ray film or with specific imaging devices.

Western blot is a reliable quantitative method only if sample properties and integrity, antibody specificity to the target protein, and loading protocols are considered.

Protein quantification is the measurement of the total protein concentration in a sample. Protein can be quantitated directly via absorbance at 280 nm or indirectly using colorimetric (BCA, Bradford, etc.), or fluorometric methods offering advantages, such as greater sensitivity.

Using equal amounts of total protein

Perhaps the most common method is the Bradford assay, which is a colour reactive assay. Other systems are available to use with samples that contain detergents, or are otherwise unsuitable for Bradford.