Week 3: Immunodetection of Western Blot for Bovine Serum Albumin (BSA) Protein

Introduction

Western blot analysis, also called the protein immunoblot or Western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract.^22,23 Western blot transfers proteins from a polyacrylamide gel onto a solid support, i.e., nitrocellulose or polyvinylidene difluoride (PVDF), which is then probed with antibodies by immunodetection to reveal target protein(s). For immunodetection, nonspecific binding sites of the membrane are first bound by excess proteins in the blocking buffer. Theoretically, any protein without a binding affinity for the target protein(s) on the membrane can be used for blocking. However, some proteins are better than others because they bind better and are more stable.²² It is crucial to select a proper blocking reagent to ensure clean and reliable results in Western blots. The two most common blocking reagents to reduce non-specific binding on membranes for Western blots are non-fat dry milk (NFDM) and Bovine Serum Albumin (BSA). The NFDM is not suitable for detecting phosphorylated proteins because it contains phosphoprotein casein, which can interact with antibodies, leading to non-specific binding and increased background noise. It may also mask some target antigens for detection if they are in low abundance, resulting in faint bands. Additionally, using NFDM can decrease the sensitivity of certain commercially available anti-His monoclonal antibodies to detect phosphorylated proteins. Thus, it is recommended to use Bovine Serum Albumin (BSA) as a blocking reagent for phosphorylated proteins to avoid the issues and gain clearer and more accurate results.

No single blocking protein or mixture of proteins works best for all cases of immunodetection of Western blots. Tests of different proteins are required to get the best possible results for a given combination of specific proteins in the blocking solutions, membrane types, and detection systems. Appropriate blocking methods are crucial to obtain clean and reliable results because they can improve the assay’s sensitivity by reducing background and increasing the signal-to-noise ratio. The ideal blocking buffer binds to all potential nonspecific binding sites to eliminate background without altering or obscuring the epitope of the target protein, which is necessary for antibodies to bind.²² Inadequate levels of blocking can result in excessive background noise and a reduced signal-to-noise ratio. However, excessive blockers may mask antibody and antigen (target protein) interactions or inhibit the marker enzyme (see below), causing a reduced signal-to-noise ratio. Each antibody-antigen pair has its unique binding characteristics. Thus, it is necessary to test different blockers for the highest signal-to-noise ratio. After completing the blocking step, the primary antibody against the antigen binds specifically to the target protein on the membrane (BSA for this lab exercise).

Antibodies (immunoglobulins) are Y-shaped proteins produced by B cells as part of the adaptive immune response when encountering a foreign molecule. Immunoglobulin G (IgG) is the most commonly used antibody type in research. There are two major kinds of antibodies based on preparations and bindings: polyclonal antibodies and monoclonal hybridomas.²⁷ Polyclonal antibodies are collected from different B cells to recognize different epitopes for the same antigen (Figure 5-8a, left and 5-8b, top). Thus, each polyclonal antibody can be used to identify and detect target proteins. In contrast, monoclonal antibodies are one IgG against a single epitope of the antigen (Figure 5-8a, right). They are prepared by repeatedly immunizing an animal, followed by isolating and fusing the animal’s spleen cells with a tumor cell line, myeloma, to form antibody-producing cells, hybridoma. The resulting cell line expresses antibodies with a single antigenic epitope (Figure 5-8b, middle).

The two traditional ways of making antibodies have drawbacks. Polyclonal antibodies produced in animals may vary among animals and bleed dates, and monoclonal antibodies from cloned hybridoma cell lines can produce more than one monoclonal antibody,²⁸ lose expression of the monoclonal antibody-encoding genes or fail to revive after cryopreservation. Therefore, recombinant DNA technologies have been used to make monoclonal antibodies (Figure 5-8b, bottom).²⁷ Scientists can use mass spectrometry to determine the amino acid sequences of the antibodies of interest and then synthesize the genes that encode those amino acids.²⁹ The heavy and light chain encoding genes are cloned into expression vectors, which can be introduced into host cells, such as bacterial, yeast, or mammalian cells, for antibody production. After subsequent purification and test for antigen binding, these antibodies can be used like those antibodies made from animals or hybridomas.

Direct vs. indirect are two ways to detect the antibody bound to the antigen (Figure 5-9). For direct detection, the antibody is conjugated to a reporter enzyme, i.e., horse radish peroxidase (HRP), alkaline phosphatase (AP), or a fluorochrome to be detected by chromogenic or chemiluminescent reactions.³⁰ Direct detection is suitable for detecting highly expressed antigens; an additional incubation step with reagents for the secondary (2°) antibody is not required. Direct detection also increases flexibility in the design of multicolor experiments if various fluorochromes are available. In contrast, a 2° antibody is raised against the host of the primary antibody and then conjugated with a reporter enzyme or fluorochrome for the indirect detection.²⁸ The primary antibody binds to the antigen and the 2° antibody. The 2° antibody is detected by being conjugated to a reporter enzyme, i.e. alkaline phosphatase. Indirect methods involve the binding of the 2° antibody, which amplifies the signal. The 2° antibody can be used with any primary antibody to which it binds due to the corresponding specificity.

In this lab exercise, we will validate the presence of the BSA protein or its homologues by immunodetection of the Western blot made in the last lab exercise. The 2° antibody used is conjugated to alkaline phosphatase, whose activity is detected by converting a colorless substrate, BCIP, in combination with NBT, to a blue precipitate (See Figure 2-7 in Module 2).

Procedure

Immunodetection of Western-blotted proteins

Note: All steps except the last two (steps 9 and 10) are conducted on a shaker at room temperature (RT).

1. Place the membrane in a plastic box slightly larger than the membrane.
2. Add the appropriate volume (~30 ml) of blocking buffer (10% bacto-peptone in 1X PBS, 0.1% Triton X-100) to the box. The buffer should cover the membrane entirely. Place the box on the shaker at low speed for 1 hr at RT.
3. Pour off the blocking buffer and wash the blot with 30 ml of washing buffer (1X PBS, 0.1% Triton-X 100) for 5 min.
4. Add 30 ml of 1:5,000 final dilution of the 1° antibody, rabbit anti-BSA Ab (1.5 mg/ml) in an antibody buffer (1% bacto-peptone in 1X PBS, 0.1% Triton X-100). Incubate for 1 hr with shaking at RT.
5. Pour off the 1° antibody solution and wash 3 x 5 min with 30 ml of wash buffer.
6. Add 30 ml of the antibody buffer containing the 1:100,000–200,000 final dilution of the 2° antibody, alkaline phosphatase-conjugated goat anti-rabbit IgG Ab. Incubate for 1 hr.
7. Pour off the 2° antibody solution and wash 3 x 5 min with 30 ml of wash buffer.
8. Incubate for 1 min with 30 ml of pre-detection buffer, and then discard the solution.
9. Add 20 ml of detection buffer (2 tablets per 20 ml RO water) and place the blot in the lab cabinet to protect it from light. Let it develop (10–30 minutes). You will see purplish blue band(s) appearing on the membrane.
10. Rinse the membrane with RO water, wrap it with Saran wrap, and store it in the refrigerator. If needed, rewetting the membrane in water can enhance the signal. This is also the ideal time to photograph the membrane for your lab report.

Data Analysis and Discussion

1. a.  How many major proteins are in the BCS/FBS sample lanes detected with last week’s (i) GelCode and  (ii) Ponceau S, as well as this week’s (iii) anti-BSA?
   2. What is the cause of the difference? Explain.
2. a.  What is the molecular weight of the main reacting protein (antigen) in the BCS/FBS sample lanes?
   2. What is its identity based on the molecular weight?
   3. Are there other bands? Explain.
3. a.  Compared to the GelCode stained gel from last week, is immunodetection more or less sensitive in detecting BSA protein? Explain.
   2. How about the specificity? Explain.
4. a.  How many major proteins in the Drosophila sample lanes appear to be recognized by the (i) Ponceau S, (ii) GelCode, and (iii) the anti-BSA?
   2. Is there any difference? Explain.
5. Do you feel confident concluding that Drosophila does not contain any protein that will react with the anti-BSA? Explain.