Explore Western blot troubleshooting video tips below, and solve some of the biggest blotting problems, such as high backgrounds, fading signals and selecting antibodies:

What is Western blotting?

Western blotting, also known as immunoblotting, or protein blotting, is a well-established and widely used detection technique for the analysis of protein molecules.
Although Western blotting protocols vary in detail among applications, with adaptations to suit specific protein characteristics and the level of information required, they all follow some common steps.

Western Blot Troubleshooting Tips:

In this guide, we’ve collated some of our best how-to video guides with helpful Western blot troubleshooting tips and techniques. From high backgrounds, to selecting the best gel, or troubleshooting your chemiluminescent detection reagent, we’ve got your Western Blot problems covered.
If we’ve missed any of your western blot troubleshooting tips, or if you have any questions, please contact us today.


How to solve problems with a high background >
Working with fluorescent >
Troubleshooting weak and fading signals >
How to obtain quantitative Western blotting results >
What to think about when selecting antibodies >
How to troubleshoot your chemiluminescent detection reagent >
How to select the best gel for your application >
How to select a transfer buffer optimal for your protein of interest >
Western blotting detection of phospho-proteins >

How to solve problems with a high background:

A high background, or a speckled background is a common problem in Western blotting applications and can be caused by too high concentration or too low concentration in your blocking agent, or by the precipitation in your blocking solution. The background can either be uneven white spots on blots, uneven dark spots and speckles on blots, or high background. This video gives you tips how to solve background problems and includes tips on filtering your blocking solution, washing your blocking agent and the various characteristics of antibodies. Watch this video to find out how to solve your western blot high background problem.

How to work with fluorescent Western blotting

Fluorescent Western blotting detection is a direct detection method where the secondary antibody is conjugated to a fluorophore, thus avoiding the need for ancillary detection reagents. The system is highly sensitive, delivers a broad linear dynamic range, and is well adapted to quantitative Western blotting. This video illustrates what the main advantages of using fluorescent detection systems in multiplex applications are, and gives tips from the Cytiva western blotting research laboratory on how to obtain the best results. Some advantages include easy normalisation of a quantitative Western blot application, as well as efficient multiplexing capability when researching two protein molecules of the same molecular size and weight at the same time. Watch this video to explore more about the advantages and how to work with fluorescent, as well as explore and learn the differences with a chemiluminescent Western blot. Click here for more information on multiplexed detection.

Troubleshooting weak and fading signals

When using a chemiluminescent system for your Western blot detection, it is common to experience weaker or fading signals after changing to a more highly sensitive detection reagent. This video explains how to resolve these signal problems, while improving your results and saving money. Some common solutions include reducing the amount of HRP in your reaction which is the result of too fast a consumption of the substrate in the detection reagent and likely caused by too high antibody concentrations – either primary or secondary, among other common issues. Watch this video to find out how to troubleshoot your weak and fading signals within your Western blot application.

How to obtain quantitative Western blotting results

In most Western blot 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. This video illustrates how you can achieve precise quantitation in Western blotting experiments, as well as the difference between qualitative and quantitative Western blotting. For additional information, watch our webinar here on quantitative western blotting.

What to think about when selecting antibodies

If you’re using a pre-stained molecular marker and can confirm the protein molecule transfer was successful, but cannot find out why your signal wasn’t received, we may just have the information. In chemiluminescent Western blot detection, too much HRP can cause too fast and too strong of a reaction, as well as substrate depletion, resulting in a quickly fading signal or non-captured signal. This video illustrates what to think about when selecting antibodies, how to save money during your research, and what to recognise when performing your Western blot.

How to troubleshoot your chemiluminescent detection

If you are experiencing problems receiving a signal in your chemiluminescent Western blot detection, the solution could be as easy as testing the functionality of your detection regent or assessing the room temperature. This video explains which tests you should perform, and provides other troubleshooting tips for chemiluminescent detection, such as not mixing the caps of the reagent pots or how to perform a blue light test or a dot blot test, among other tips.

How to select the best gel for your application

In Western blotting, the type of gel you should use for electrophoresis, as well as the acrylamide concentration in the gel, varies with each application. Watch this video to explore how to select the best gel for your application that will give you the best results for the protein or proteins molecule(s) you are testing, such as what is the best acrylamide concentration to the appropriate molecular weight.

How to select a transfer buffer optimal for your protein of interest

When it comes to Western blotting transfer buffer selection, concentration of methanol and sodium dodecyl sulfate (SDS) are key parameters for transfer efficiency. These reagents affect the solubility of the protein as well as its ability to bind to the membrane. This video discusses using a recommended transfer buffer, the purpose of methanol in a transfer buffer and how to adjust the buffer composition to improve transfer efficiency of your protein of interest. Additionally, for more information, click here to read our guide on how to set up a Western blot wet transfer.

Western blotting detection of phospho-proteins

The buffers and blocking agents you use to detect phospho-proteins can have a big impact on your results, especially using commercially available dried milk as a blocking agent. Watch this video to find out why and explore our troubleshooting tips on how to obtain the best results when detecting phosphorylated proteins by Western blotting, such as why you should use a TBS buffer instead of a PBS buffer or how to avoid high backgrounds in your Western blot analysis.