western blot百替生物

Chem/Biol 474 February 2007

WESTERN BLOT

INTRODUCTION

There are three parts to this experiment. First, proteins will be electrophoretically separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) (Weber and Osborn, 1975). Second, the proteins will be electrophoretically transferred from the gel to nitrocellulose. Third, the nitrocellulose will be analyzed for the presence of a specific protein using an enzyme immunoassay (EIA).

SDS-PAGE

Consult the prior electrophoresis experiment for background information on SDS-PAGE.

BLOTTING

Nitrocellulose (NC) membranes, the standard medium for protein transfer techniques, remain the most widely used and versatile supports for this application due to their high affinity for proteins and compatibility with a variety of assay conditions and detection methods. They are also simple to "block," i.e., to treat to prevent non-specific binding, in comparison to other types of solid supports. The mechanism of protein binding to the membrane is most likely a hydrophobic interaction. Proteins can be removed from the membrane with non-ionic detergents, and binding in most transfer buffers occurs when both the NC surface and the protein molecule are negatively charged, thus eliminating the possibility of an electrostatic interaction. The membranes are cast under controlled conditions to achieve an even pore size and uniform, consistent binding.

Blotting was first performed by Southern in 1975 with the transfer of DNA from agarose gels to nitrocellulose membranes. Since that time, blotting has been applied to RNA and protein from both agarose and polyacrylamide gels. To circumvent the

inefficiencies observed in various capillary transfers, electric current was used as the force in eluting protein from polyacrylamide gels, as first described by Towbin et al., (1979a). Since that time, the use of electrophoretic transfer has been applied to both DNA and RNA. Numerous publications have dealt with the topic of protein electrophoretic transfer technique (e.g., Lin and Kasamatu, 1983). There has also been a review summarizing the expanding literature being generated on electroblotting methodology (Gershoni and Palade, 1983).

Western blotting involves the electrophoretic separation of proteins followed by electrophoretic transfer to nitrocellulose. At this point the proteins are on a solid matrix ready for immunodetection. This provides a powerful tool for identifying specific proteins in a complex mixture since only proteins recognized by the first antibody will lead to eventual color development (Towbin, et al., 1979; Reiser and Wardale, 1981). For example, in the Western blot test for individuals carrying antibodies to the human immunodeficiency virus (HIV) that causes AIDS, viral proteins are separated by electrophoresis followed by transfer to nitrocellulose. A positive test will depend on the binding of specific antibodies in blood serum to these proteins. The final step is accomplished with horseradish peroxidase (HRP) attached to antibody that recognizes the previously used antiviral antibody.

IMMUNOLOGY

Proteins carry out several types of biological functions including transport and storage, muscle contraction, mechanical support, immune protection, generation and transmission of nerve impulses, and the control of growth and differentiation. When a protein performs a catalytic function, it can be assayed by a suitable choice of substrate coupled with a method of following the change in substrate or product concentration with time. However, this cannot be done when the protein is not an enzyme, and the assay of noncatalytic proteins may be difficult. Fortunately, the field of immunology provides a powerful method for detecting specific noncatalytic proteins.

Vertebrate animals have developed a specialized network, called the "immune system", for combating foreign pathogens that might enter their circulatory systems and/or organs. The immune system is divided into two branches, the humoral system and the cell-mediated system. In the humoral immune system, small lymphoid cells called B-cells (because they originate and mature in the bone marrow), are stimulated by a foreign invader (the antigen, ag) to produce specific glycoproteins (antibodies, ab, or immunoglobulins, Ig) that bind to and effectively neutralize the antigen.

Antibody molecules come in 5 different classes: IgM, IgG, IgD, IgE and IgA. These different classes of antibodies have different specific functions e.g. IgA is found in mucosal secretions whereas IgG is the most common antibody in blood. Although the effector functions of the different antibody classes vary, they share a common structure. The basic unit of an antibody molecule consists of 2 identical light chains (ca. 25,000 in MW) and 2 identical heavy chains (MW = ca. 50,000). The 4 subunits are connected by disulfide bonds and the overall appearance of the antibody molecule is that of a Y shape. The arms of the Y are the antigen binding sites, and hence are variable. The base of the Y is conserved among different antibodies and is called the constant region.

The term γ-globulin refers to an electrophoretic fraction containing IgM, IgG, IgD, IgE, and IgA.

Fig 3-1 Diagram of the human IgG molecule: first version. Shaded bars indicate polypeptide chains, -S-S- lines disulphide bonds, dotted lines places of enzymatic cleavage, and number indicte amino acid positions. C, constant region; CHO carbohydrate; COOH, carboxyl end; H, heavy chain; L, light chain; hv, hypervariable reigon; NH2, amino end; V, variable region. (Modified from Klein, J. (1982) Immunology -

The Science of Self-Nonself Discrimination. Wiley, New York.)

In an EIA, (enzyme-linked immunosorbent assay) the antigen is first bound to a nitrocellulose membrane. Following binding of antigen, the remaining unbound sites on the nitrocellulose are blocked with a protein that will not bind to antigen. Membrane-bound antigen is then incubated with an antibody specific for the antigen to be detected, and the membrane is washed to remove unbound antibody. This washing is followed by the addition of a second antibody generated against the constant region of the first, which therefore combines with the first. The second antibody contains a covalently attached enzyme which can be detected by an appropriate assay.

In this experiment, the antigen is myoglobin. The myoglobin was injected into rabbits to produce antibodies.

As shown in the diagram above, Ag binds the arms of the Y-shaped Ig molecule. The second antibody was produced by injecting rabbit IgG into goats and is called goat anti-rabbit (GAR) antibody. The antibody produced was purified and then covalently labeled with an enzyme called horseradish peroxidase (HRP). Since goat anti-rabbit antibody binds to the antigenic determinants on both heavy and light chains of the rabbit antibody, it can be used to detect any antigen-rabbit IgG complex.

Horseradish peroxidase catalyzes the following oxidation-reduction reaction between 4-chloro-1-naphthol and hydrogen peroxide:

The 1,4-naphthoquinone produced in this reaction is purple which allows detection of the presence of the initial antigen.

High grade reagents must be used in this experiment to avoid problems with impurities that might interfere with the analysis. Azide ion is especially troublesome since it is an inhibitor of horseradish peroxidase.

EXPERIMENTAL

SDS-PAGE Solutions

Prepared by the prepper

1. Myoglobin (10 mg/mL)

Dissolve 100 mg myoglobin in 10 mL high resolution buffer (HRB) at pH

8.8. (Gelman Cat. No. 51104).

2. Lysozyme (10 mg/mL)

Dissolve 100 mg lysozyme in 10 mL high resolution buffer (HRB) at pH

8.8.

(Gelman Cat. No. 51104)

3. High resolution buffer at pH 8.8 contains tris (hydroxymethyl)

aminomethane, barbital, and sodium barbital.

4. Coomassie Stain: Mix 50 mL isopropanol, 50 mL acetic acid, 150 mg

CBB-R (Coomassie Brilliant Blue R), 150 mg cupric acetate, and 400 mL

distilled water. Stir the staining mixture for several hours. *Use a

replaceable container for mixing the staining solution as the color is not

easily removed.*

5. Destain: 10% glacial acetic acid, 10% methanol.

Loading onto the gel

Boil all samples before loading onto a 15% (w/v) acrylamide gel.

Staining and analysis

After electrophoresis and before opening the gel sandwich record kaleidoscope colors and their position on the gel.

IMPORTANT: After the electrophoresis cut the gel on the dye front. Also cut between the appropriate lanes. The left side will be stained with Coomassie and the right side will be used for blotting.

Transfer the left gel fragment to a pan partly filled with STAINING SOLUTION (Coomassie Blue). Add more STAINING SOLUTION if necessary to completely cover the gels. The gels are left in the STAINING SOLUTION on a rotary shaker overnight. The next morning your instructor will pour off the STAINING SOLUTION (recycle) and add destaining solution. The pan will be placed back on the shaker. Including a piece of foam rubber or Kimwipes in your destaining pan will increase the rate of destaining.

BLOTTING (Semi Dry Protocol)

Dunn Transfer Solution:

10mM NaHCO3

3 mM Na2CO3

20% methanol

pH 9.9 approx

dissolve: 0.84 g NaHCO3

0.318g Na2CO3 in dd H2O

Add 200 mL methanol

Adjust to 1 L w/ ddH2O

NOTE: do not adjust pH. Store at 4°. Use approx 150 mL of buffer for each transfer.

Loading:

NOTE: Remember to wear gloves in order to avoid contamination of the blot. Soak four pieces of filter paper, one nitrocellulose membrane and the protein gel in transfer buffer for 15 min. Make a sandwich w/two pieces of filter paper, nitrocellulose, gel, and two pieces filter paper, in that order, on the surface of the blotter. Smooth and press out bubbles with a pipet. Make sure the sandwich is saturated with solution. Place top plates in place, taking care not to disturb/shift the sandwich.

--filter paper

filter paper

gel

nitrocellulose

filter paper

+filter paper

NOTE: This is a downward transfer.

Run the transfer at a constant current of 200mA for 8 hours. Be sure to thoroughly clean and rinse the unit after each run.

After the transfer, air dry the nitrocellulose. Then spot the following controls: 2 μL of 10 μg/μL myoglobin and 2 μL of 10 μg/μL lysozyme. Redry the nitrocellulose.

ENZYME IMMUNOASSAY

Equipment

1. Platform shaker

2. Cover a 10 inch x 10 inch piece of styrofoam with aluminum foil since this provides

a clean and resilient surface for the nitrocellulose membrane.

3. Continuously adjustable digital micropipettes such as P20 and P200 Gilson

Pipetman are needed.

Reagents

1. Deionized water (d H2O) should be used throughout this experiment.

2. The first antibody, rabbit antiserum to myoglobin.

3. The second antibody, goat anti-rabbit IgG(H+L) horseradish peroxidase conjugate,

is available from Bio-Rad (Cat. No. 170-6515).

4. Blocking Solution contains 6% (w/v) dried milk in TBS.

5. Tris buffered saline (TBS) contains 20 mM tris (hydroxymethyl) aminomethane and

500 mM NaCl at pH 7.5. To prepare TBS add 4.84 g of Tris to 58.45 g NaCl and bring to approximately 1800 mL with dH2O. Adjust to pH 7.5 with HCl and then

dilute to a total volume of 2.00 liters.

6. Tween Tris-buffered saline (TTBS) is prepared by adding 0.5 mL tween-20 (EIA

grade, Bio-Rad Cat. No. 170-6531) to 1.00 liter of TBS.

7. Horseradish peroxidase (HRP) color development reagent can be purchased from

Bio-Rad (Cat. No. 170-6534).

EIA Procedure

Label a 50 mL Erlenmeyer flask on the bottom with your name and then completely cover the flask with aluminum foil. Place this in the freezer for later use in color development.

In this and all subsequent steps, it is important to submerge the filter rather than having it float on the surface of the solution. Immerse it at a 45° angle.

Fill a petri dish with 40 mL of blocking solution. Immerse the membrane into the blocking solution and swirl on a platform shaker for 30 minutes. Follow this by two 5 minute washes in 40 mL TTBS with gentle agitation.

First antibody: Place 14 mL blocking solution plus 28 mL Tris buffered saline (TBS) in a dish. Immediately prior to use, add 42 m L of the antibody. Then transfer the filter from the blocking solution into the first antibody solution and swirl for 1 hour. Wash in 40 mL Tween Tris-buffered saline (TTBS) for 5 minutes with swirling. Repeat twice more with fresh 40 mL TTBS for another 5 minutes with swirling.

Second antibody: Just prior to use, add 14 m L of the second antibody to a petri dish containing 14 mL blocking solution and 28 mL TBS. Transfer the filter to the second antibody solution and swirl for 1 hour. After that time, rinse in TTBS for 15 minutes. Then do a repeat wash in 40 mL TTBS for 5 minutes with swirling. Wash in 40 mL of TBS to remove detergent prior to color development.

Color development: Prepare the following solutions:

1. CAUTION: Wear gloves to avoid contact with the HRP reagent. Dissolve 24

mg HRP reagent in 8 mL ice-cold methanol in a 20 mL beaker. Wrap with

aluminum foil since the reagent is light sensitive.

2. Remove a cold Erlenmeyer flask from the freezer. Add 40 mL TBS followed

by 24 m L ice-cold 30% H2O2 with mixing.

Mix solution 1 and 2 immediately before use and then transfer to a petri dish. Transfer the filter to the petri dish and allow to incubate at room temperature for 15-30 minutes. Stop the development by immersing the filter in dH2O for 10 minutes, changing the water at least once. Dry the membrane suspended on pins.

DATA

1. Store each side of gel in a separate Seal a Meal and then mount on 8 1/2 x 11 inch white paper and submit with your lab report. Do not add liquid to Seal a Meal pouch. Also, include a photocopy of Coomassie-stained gel and the actual gel.

2.

Store nitrocellulose from the EIA procedure in a separate Seal a Meal and then mount on 8 1/2 x 11 inch white paper and submit with your lab report.

RESULTS

1. From the Coomassie stained gel measure the migration distance for Kaleidoscope protein standards and complete the following table:

The following shows how to calculate Rm:

dye

x

m d d R = 2. Graph log (molecular weight) on the vertical y-axis versus R m on the horizontal x-axis. Draw a straight line through your data points.

3. Show on the photocopy the identification of protein bands by color and

composition. Use an arrow to show the migration direction.

4. Estimate the molecular weight of myoglobin from the graph.

How well does your experimental value compare with the published literature

value? Cite your reference.

5. Calculate m g of each protein loaded into each well. Note that Kaleidoscope

concentration is 1.6 mg total protein in 500 m L. Assume that the 7 Kaleidoscope proteins contribute equally by mass.

DISCUSSION

1. Compare the specificity of Coomassie stain to the specificity of the EIA procedure. Compare the bands seen by Coomassie stain and by enzyme immunoassay. Discuss the origin of all bands seen in the Western Blot.

2. Discuss the purpose of spotting myoglobin onto the nitrocellulose (positive control).

3. Discuss the purpose of spotting lysozyme onto the nitrocellulose (negative control).

QUESTIONS

You should refer to specifics in the following questions. The EIA procedure involves 1°Ab, 2°Ab, HRP, H2O2, and 4-chloro-1-haphthol.

1. What would you conclude if a myoglobin band appeared on the Coomassie

stained gel but there was no spot for the post transfer EIA positive control? List possible causes of the problem.

2. What would you conclude if lysozyme appeared on the Coomassie stained gel

and also gave a spot for the EIA negative control?

REFERENCES

BLOTTING

1. H. Towbin et al., Proc. Nat. Acad. Sci., 76, 3116 (1979a).

2. H. Towbin, G. Staehlin, J. Gordon, Proc. Nat. Acad. Sci., USA, 76, 4350-4354

(1979b)

3. W. Lin, and H. Kasamatu, Anal. Biochem. 128, 302 (1983).

4. E.M. Southern, J. Mol. Bio.98, 503 (1975).

5. J. Reiser, J. Wardale, Eur. J. Biochem.114, 569-575 (1981).

6. J.M. Gershoni, and G.E. Palade, Anal. Biochem. 131, 1 (1983).