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Step 2 – Electro-Transfer
Western Transfer System Transfer Buffers and Membranes Protein Stain Kits Signal Enhancement
Step 3 – Blocking
Introduction Blocking Buffers
Thermo Scientific Pierce Western Blotting Handbook and Troubleshooting Guide
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Substrate
Detectable Product
Substrate
Detectable Product
Enzyme
Enzyme A. B.
Direct Detection Figure 1.
Indirect Detection
Figure 1A. In the direct detection method, labeled primary antibody binds to antigen on the membrane and reacts with substrate, creating a detectable signal. 1B. In the indirect detection method, unlabeled primary antibody binds to the antigen. Then, a labeled secondary antibody binds to the primary antibody and reacts with the substrate.
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The first step in a Western blotting procedure is to separate the macromolecules using gel electrophoresis. After electrophoresis, the separated molecules are transferred or blotted onto a second matrix, generally a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. Next, the membrane is blocked to prevent any nonspecific binding of antibodies to the surface of the membrane. The transferred protein is complexed with an enzymelabeled antibody as a probe. An appropriate substrate is then added to the enzyme and together they produce a detectable product such as a chromogenic precipitate on the membrane for colorimetric detection. The most sensitive detection methods
Step 4 – Primary Incubation
Primary Antibodies 24 Custom Antibody Services 25 Epitope Tag and Loading Control Antibodies 26–27ຫໍສະໝຸດ Step 7 – Wash
Dry Buffers 48
Step 8 – Incubation with Substrate
Chromogenic Substrates for HRP 49–50 Chromogenic Substrates 50 for Alkaline Phosphatase Chemiluminescent Substrates 51–52 Standard Chemiluminescent 53–59 Western Blotting Substrates and Kits Fast Western Blotting Kits 60–61 Specialized Western Blotting Kits 62 Far-Western Blotting Kits 63 64 65–68
use a chemiluminescent substrate that, when combined with the enzyme, produces light as a byproduct. The light output can be captured using film, a CCD camera or a phosphoimager that is designed for chemiluminescent detection. Whatever substrate is used, the intensity of the signal should correlate with the abundance of the antigen on the blotting membrane. Detailed procedures for detection of a Western blot vary widely. One common variation involves direct vs. indirect detection (Figure 1). With the direct detection method, the primary antibody that is used to detect an antigen on the blot is labeled with an enzyme or fluorescent dye. This
Step 10 – Stripping
Film 69 Background Eliminator 70 Stripping and Reprobing a Membrane 71 Stripping Buffers 71–74 Optimize the Signal-to-Noise Ratio 74 Protocol for Stripping an Immunoblot 74–75
Troubleshooting Guide
Optimizing Chemiluminescence Blotting 76 Optimizing Antibody Concentration 77 Optimizing Antigen and Antibody Concentration 78 Problem Guide 79–82 Full-Length Western Blotting Protocol 83 Using Chemiluminescent Substrates
Table of Contents
Western Blotting Overview
Western Blotting 10 Steps Overview 2–3 4–5 6 7–12 11–12 13 14–15 15–17 18–19 20–23
Step 5 – Wash
Washing the Membrane Dry Buffers 28 28 29 30 30–31 32 33–37 38–40 41 42–43 44 45–47
detection method is not widely used as most researchers prefer the indirect detection method for a variety of reasons (Table 1). In the indirect detection method, a primary antibody is added first to bind to the antigen. This is followed by a labeled secondary antibody that is directed against the primary antibody. Labels include biotin, fluorescent probes such as fluorescein or rhodamine, and enzyme conjugates such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). The indirect method offers many advantages over the direct method (Table 2).
Step 9 – Target Detection
Introduction and Troubleshooting Digital Imaging and Software
Recommended Reading
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Western blotting overview
The term “blotting” refers to the transfer of biological samples from a gel to a membrane and their subsequent detection on the surface of the membrane. Western blotting (also called immunoblotting because an antibody is used to specifically detect its antigen) was introduced by Towbin, et al. in 1979 and is now a routine technique for protein analysis. The specificity of the antibody-antigen interaction enables a target protein to be identified in the midst of a complex protein mixture. Western blotting can produce qualitative and semiquantitative data about that protein.