How To Read a Western Blot: A Guide to Understanding Results

Introduction

Western blotting is a powerful technique used in molecular biology and proteomics labs to study proteins obtained from a cell lysate or other protein samples. It’s a powerful procedure to analyze the relative mass, presence, and relative abundance of post-translational modifications in proteins. Further, it’s also a crucial tool to study protein-protein interactions.

The technique was first introduced by Towbin et al. and Burnette. It’s based on the interaction between target antigens of a protein sample with specific antibodies.

The workflow involves first separating the proteins based on their molecular weight (in kDa) in the form of bands through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transferring the bands on the nitrocellulose membrane or PVDF membrane using electric current (the process known as immunoblotting).

The target protein bands are then incubated with the primary antibody having epitope for the protein of interest followed by a secondary antibody conjugated with an enzyme or fluorescent molecules. This helps in the detection of proteins.

Today, many detection methods of proteins are available, such as chemiluminescence, fluorescence, and calorimetric detection techniques. Further, the conjugates’ most frequently used detection techniques include Alkaline Phosphatase (AP) and Horseradish Peroxidase (HRP).

In this article, you’ll learn in detail about the stages of western blotting, how to produce the results, and read the western blot results

The Stages of Western Blotting

The western blotting assay is mainly composed of seven main steps:

1. Sample Preparation: Cell or tissue lysate of your choice of the cell line, or any other protein sample of choice is prepared to isolate and analyze specific proteins and loaded on SDS-PAGE.
2. Electrophoretic separation of Proteins: The total protein samples are separated based on their molecular weight by using gel electrophoresis. Then, the obtained protein bands are electronically transferred onto a nitrocellulose or polyvinylidene difluoride (PVDF) membrane — the western blot technique.  
3. Block non-specific sites to prevent non-specific binding: To prevent non-specific binding of antibodies to the target protein of the membrane, the immunoblot or the membrane is blocked using the blocking buffer (generally 5% BSA, prepared in dilutions of TBST). The membrane is then washed with a wash buffer (generally tris buffered saline, TBS, or phosphate-buffered, saline, PBS, with 0.1% tween 20) to remove any stuck residue.
4. Incubation with protein-specific antibodies: Primary antibody specific to the protein of interest is used to incubate with the membrane followed by incubation with an enzyme that conjugates a secondary protein specific to the primary protein. The antibodies commonly used in the process are either monoclonal or polyclonal antibodies.
5. Target protein detection: This step involved visualization of the target protein-antibody-antibody complex. A substrate known as ECL combines with the enzyme conjugated to the secondary antibody.
6. Imaging: It’s the last step of the western blotting technique where a charged-coupled device (CCD) camera or scanner collects emitted light to capture the image of the target protein from the membrane.

Result Analysis: The image process results in the formation of target protein bands that are analyzed to determine the amount of protein in a sample or protein concentration and study protein expression.

How to Read a Western Blot

Reading the results after the Western Blot protocol is one of the crucial steps that one needs to carefully perform. If you don’t read the results accurately, expensive reagents or high-tech equipment used in the experiment will be of no use. It’ll also spoil the time and effort put into the process.

The identity of proteins is confirmed by comparing the molecular weight markers (for size) and positive control. The intensity of the protein band helps in the quantification of the protein of interest in the sample. While many applications employ the technique to determine the presence and concentration of protein, in some cases, it’s used to define protein levels in relative or absolute terms using quantitative analysis of proteins.

Quantitation

• Identify the linear range: Understanding the linear range is essential to accurately interpret your data. The linear range is defined as the range of signal intensities where the concentration of the target on the membrane is linearly related to the signal intensity recorded by the detector. Outside the linear range, the amount of protein won’t have a linear relationship with the band intensity.
• Let’s understand this from a graph:

In the above diagram, you can observe two regions: the toe region and the shoulder region. The toe region indicates low sensitivity where sample concentration may not be reflected in the signals. And, the shoulder region shows a strong signal, where data is saturated and which may alter data analysis.

What is saturation?

Saturation represents a point at which even after increasing the protein concentration doesn’t bring any changes in the band intensity. At saturation, you may lose data. Thus it’s necessary to identify the linear range accurately.

To avoid saturation on film, you need to perform the dilution of your amount of protein lysate or sample, use lesser antibody concentration, and reduce film exposure length.

• Remove Background noise: You must have observed a major problem in western blot analysis is the high background noise, such as one side of the blot being darker than the other side. It causes inconsistencies in your results. The issue can be solved using some software packages that calculate the background around the target protein.

Normalization

Normalization in western blotting is analyzing and comparing the bands of a loading control (an abundant protein, which is not expected to change during the experiment) to the bands of specific proteins across the blot. The step is essential because even errors in western blots are unavoidable. It can be due to pipetting errors, inconsistent sample preparations, and uneven protein transfer.

The control proteins used for normalization are known as housekeeping genes, such as beta-tubulin, actin, and a chaperone protein like Hsp70. It’s assumed that the expression level of these proteins will remain the same during the experiment. However, you may also observe some level of inconsistencies with these proteins too.

Acquire Western Blot Equipment with Excedr

Western blot analysis is a crucial and effective tool to study all about proteins, ranging from their expression, concentration, and post-translational modifications. It consists of almost seven stages that include phases from protein isolation, blotting to the membrane, imagining, to the analysis of the result obtained.

After performing the experiment, analyzing and reading the result accurately is the next crucial step that needs to be carefully performed. What needs to be taken care of at this phase is correctly identifying the linear range, reducing background noise, and proper normalization of bands.

To deduce results correctly, you need to obtain clear images and data which you can easily interpret. This demands researchers to use high-quality reagents and high-throughput equipment to be employed in the process. However, it can be heavy on your pocket and might cause you to break the bank.

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