Introduction to Western Blotting
In this learning path, you will learn about the science, best practices and in-lab procedures behind each step in the Western blot workflow from preparing your samples through collecting your data. Happy blotting!
- 11 courses
- 83 lessons
- Lifetime access
- Certificate of completion
What is a Western Blot?
Western blots are used a several different areas of life science research. This course will dive into the process of a Western blot, as well as why and when you would choose to use that technique.
Preparing the Samples
Your Western blot can only be as good as the samples you start with. Learn more about the things to consider when preparing your samples and how to minimize the amount of variability that gets added to your experiment.
Preparing the Gel
It is important to take your targets into consideration when selecting the gel type and percentage, as well as buffer composition when running and SDS-PAGE. This will help ensure that you have an even run of your samples, which will impact the accuracy and robustness of your data.
Equilibrating the Gel and Preparing for Transfer
Gel equilibration is a critical step to help ensure an even transfer of your proteins from the gel to the membrane. It's also important to choose the right membrane for your targets. Paying close attention in these details will help increase the accuracy and robustness of your data downstream.
Transferring the Proteins
Protein transfer is a distinguishing step of the Western blotting protocol. It is important to consider all of the aspects of transfer including your transfer conditions, buffer, and apparatus. These have the potential to impact your transfer, and ultimately your Western blot data.
Drying the Membrane
Drying the membrane may sound odd, but it is actually better for your Western blot data in the long run. In this course, we will discuss why you should dry your membrane, and how to accomplish that to leave you with a great Western blot membrane.
Chemiluminescence: Blocking the Membrane
Chemiluminescence is a commonly used detection method for Western blotting. In this course, you'll learn about the blocking step in the chemiluminescent Western blot protocol and how you can reduce variability in your results by selecting the right blocker for your experiment.
Chemiluminescence: Primary Antibody Incubation
Learn the science behind how primary antibody incubation works, and why it is an important step in the process. You will also learn some tips that you can implement in your workflow to continue getting robust data from your blots.
Chemiluminescence: Secondary Antibody Incubation
Secondary antibodies can have a large impact on the quality of data that you get from your Western blots. Learn about the role that secondary antibodies play in the Western blot process, and some best practices for getting great results.
There are several things to keep in mind when using a chemiluminescent detection system. In this course, you'll learn about the importance of the substrate and how it could impact your final results, as well other considerations that will help you get consistent and robust data from your Western blo
Blocking Step Through Image Adjustment for NIR
Learn about the basics of Near-Infrared Western blotting, including blocking the membrane, antibody selection, and more.
Dilutions and Calculations
Check this course out for a refresher on the basics of dilutions and other calculations needed for Western blotting.
Introduction to Quantitative Western Blotting (FREE)
Learn what a quantitative Western blot is and is not, and how it differs from a qualitative Western blot.
Quantitative Western Blot Analysis with Replicate Samples
Replicate samples are essential to quantitative Western blotting. Learn more about technical versus biological replicates, and how to incorporate replicates into your experiments.
Publishing Western Blot Data
Learn more about the things to consider when working towards publishing your research, including the peer review process and the importance of choosing the right journal.
Data Analysis and Basic Quantification
Proper data analysis and quantification are essential when drawing meaningful conclusions from your data. Improving in these areas will have a significant impact on your research.
Linear Range for Quantitative Western Blots
Learn more about what linear range and dynamic range are, why they are important, and how they play a significant role in getting high quality data that you can count on.
There are several things to consider before beginning your experiments. Learn more about the importance of normalization and how it can be done using three different strategies.
Antibody validation is an important step in the Western blot process. Learn more about how specificity, selectivity, and reproducibility impact your antibody choice.
Antibody Ins and Outs
Learn about the importance of antibodies and how their different qualities can impact your experiments.
Antibody selection is an important step in ensuring the antibodies in your experiment give you reliable results.
Presentation: How to Best Present a Quantitative Western Blot for Publication
Hear from Deputy Editor, F. Peter Guengerich, Ph.D, about The Journal of Biological Chemistry’s revised guidelines for presenting your quantitative Western blots and improving the reliability of research.
Estimating Protein Concentration
Learn about the importance of estimating your protein concentration and how to incorporate this technique into your experimental design.
The BCA assay is one of the most popular protein estimation assays. Learn how to perform this assay and how you can integrate it into your workflow.