Western Blotting

Enhance your Western blot workflow with this in-depth training course. Learn how to design, perform, and analyze Western blots confidently for reliable protein detection and quantification.

Contents

Introduction

Welcome to Proteintech's Western Blotting online training course.

In this course, we will teach you how to perform and optimize each step of a western blot - everything from sample prep to image analysis.

This course is designed to be completed in 2-3 hours. Your progress will be saved after each lesson so you can easily return and continue when you're ready.

Remember to mark each section as "complete" using the button in the bottom right corner once you've finished.

Let's get started!

Western Blot Introduction
Western Blot Protocol

Sample Preparation

Optimizing your sample preparation is crucial for obtaining accurate results. It is an often overlooked part of the protocol that is highly dependent on your sample type and protein of interest.

In this chapter, we will learn how to lyse different sample types and how to optimise this vital step in the western blot workflow.

Lysate Preparation
Lysis Solution
SDS Sample Buffer
Heat Denaturation
Quiz - Sample Preparation

SDS-Page

SDS-PAGE is a technique widely used to resolve proteins from a mixture according to their size. SDS coats the proteins in a uniform negative charge, ensuring their migration is solely dependent on molecular weight.

In this chapter, we will teach you how to optimize your gel electrophoresis for high quality protein resolution.

SDS-Polyacrylamide Gel Electrophoresis
Gel Layers and Recipes
Gel Loading

Transfer

Now that you have resolved your protein on SDS PAGE, you’re going to want to transfer the newly resolved proteins onto a solid support so you can detect your specific proteins of interest with their respective antibodies.

Transfer Methods
Membrane Choice
Quiz - SDS-Page and Transfer

Immunoblotting

Once the proteins have been transferred to a membrane, we can now identify our protein of interest using specific antibodies.

Blocking and Optimizing for High Background
Choosing the Primary Antibody
Primary Antibody Optimization
Choosing the Secondary Antibody
Western Blot Controls
Washing
Quiz - Immunoblotting

Detecting and Analyzing Your Results

Now that you have performed your Western blot, it is time to detect and analyze the data you've produced.

Chemiluminescence Detection
Fluorescent Detection
Colorimetric Detection
Results Interpretation

Advanced Western Blot Tips

Western blot is an incredibly varied field, with a vast amount of special cases and sub-techniques. This section of the course will cover some of the more common special cases and give you some helpful tips and tricks to get you started!

Phosphoproteins
Small Protein Tips (<20 kDa)
Large Protein Tips (>100kDa)

Western Blot Troubleshooting

This chapter will help you identify specific problems with your Western blot and provide you with the knowledge to troubleshoot.

Extra Bands
High Background
Faint Bands / Weak Signal
High Lipid Content
High Lysate Concentration
Long Term Sample Storage
Quiz - Troubleshooting

Summary

Congratulations on completing the Proteintech Learning Portal Western Blotting course. You are now ready to start detecting your proteins!

Let's recap what we have learned.

Technical Guide
Take-home Messages