REVISIÓN DEL DISEÑO

specific antibody followed by chemiluminescent detection methods. Major advantages of this method include the technical ease of preparing the gel, increased sensitivity, and a much shorter turn-around time than in-gel antibody analysis (see Alternate Protocol). It also eliminates the use of radioactive isotopes. In addition, the primary and secondary antibodies can be easily removed and the membrane (PVDF) probed again with a different detection antibody. This allows the laboratory the opportunity to detect a second protein or antigenic site on the same protein and also a “second chance” to correct an omission in the procedure, especially for samples in limited supply (UNIT 6.2); however, the immunoblotting step of such large proteins must be done with care in order to assure the adequate transfer of the largest proteins. The Alternate Protocol uses direct identification of the protein in the gel by a specific radiolabeled antibody, eliminating the immunoblot- ting step, but sacrificing sensitivity.

BASIC PROTOCOL

AGAROSE GEL ELECTROPHORESIS AND BLOTTING WITH IMMUNODETECTION

The following is a method for separating a complex mixture of human plasma proteins by continuous SDS horizontal (submerged) agarose gel electrophoresis. The very large multimers of circulating plasma von Willebrand factor (or more highly purified prepara- tions of this protein) are identified using a specific antibody and visualized using a chemiluminescent reagent. This protocol utilizes a 20 × 25–cm horizontal gel apparatus. Dry agarose is weighed, mixed with electrophoresis buffer, and melted in a hot water bath. The agarose is allowed to partially cool, then is poured into a horizontal casting frame with a Teflon comb in place, and allowed to solidify. The gel is covered with 1 to 2 mm cold electrophoresis buffer and the comb is carefully removed. Prepared samples contain- ing the proteins of interest are diluted with sample buffer and loaded into wells. Electro-

phoresis is carried out for 3 to 6 hours at 4°C. The gel is placed into a vertical tank transfer Electrophoresis_and Immunoblotting

apparatus (UNIT 6.2), and the proteins are transferred overnight onto an immobilization matrix (PVDF membrane). The membrane is blocked and probed with a polyclonal primary antibody specific for the antigen of interest and, after washing, a conjugated secondary antibody is introduced. After incubation the membrane is washed again, and the protein bands of interest are illuminated with a chemiluminescent detection system. Materials

SeaKem HGT(P) agarose (Bio Whittaker or equivalent) 1× electrophoresis buffer, 4°C (see recipe)

Protein samples

2× sample buffer (see recipe)

0.25× transfer buffer without methanol (see recipe)

Blocking buffer (UNIT 6.2) containing 5% (w/v) nonfat dry milk, fresh Antibodies:

Primary: rabbit anti-vWF (Dako)

Secondary: donkey horseradish peroxidase–linked anti-rabbit Ig (Amersham Pharmacia)

ECL Western Blotting Analysis system (Amersham Pharmacia) Aluminum foil

Boiling waterbath (optional)

Horizon 20-25 horizontal electrophoresis apparatus (Life Technologies) or equivalent

Teflon comb (e.g., 1 × 9–mm, 20-well) Pipet with fine tip or equivalent

0.45-µm Immobilon-P polyvinylidene fluoride (PVDF) membrane (Millipore) Additional reagents and equipment for protein transfer to membranes and

immunoblotting (UNIT 6.2) Cast agarose gels

1. Weigh 1.2 g SeaKem HGT(P) agarose and transfer to a 250-ml flask containing 200 ml of 1× electrophoresis buffer. Add a Teflon-coated magnetic stir bar and tightly cover the flask with aluminum foil.

2. Heat in a boiling water bath, with slow mixing to avoid bubbles, until clear. Alterna- tively, microwave until boiling

CAUTION: Whenever a solution is heated in a microwave the chance of superheating

(“boil up”) is always present. Protective gloves, gown, and eyewear should be worn at all times.

3. Assemble Horizon 20-25 horizontal electrophoresis apparatus or equivalent accord- ing to manufacturer’s recommendations.

The wedge shaped casting dams of the Horizon (Life Technologies) horizontal apparatus are easily placed in backwards, resulting in leaking of liquid agarose. Care should be exercised so that the dams form a perpendicular angle with the UVT tray.

4. Cool agarose to 55° to 60°C and pour the molten agarose into the electrophoresis apparatus to a depth of 4 mm.

It is often helpful to prewarm the electrophoresis apparatus with warm water or in an oven to avoid cooling the agarose and causing the agarose to solidify unevenly.

5. Carefully insert the desired (e.g., 1 × 9–mm, 20-well) Teflon comb with care to avoid bubbles.

Agarose Gel Electrophoresis of Proteins

Removing the comb while the agarose is still in a molten state and then reinserting often eliminates the formation of bubbles under the teeth of the comb.

6. After the agarose has solidified, place the apparatus at 4°C and allow the agarose to age 20 to 30 minutes.

The electrophoresis is to be carried out at 4°C. The apparatus may either be placed in a cold room, the electrophoresis buffer can be circulated through a refrigeration unit, or the apparatus can be packed in wet ice.

7. Overlay the solidified gel with 2 to 3 mm electrophoresis buffer, 4° C. 8. Remove the sample comb by lifting vertically in one smooth motion.

It is helpful to hold the gel down with the gloved fingers of the other hand to keep the gel from being pulled up when the comb is removed.

Prepare and load sample

9. Dilute the protein samples to twice the desired concentration using distilled water. Immediately add the diluted sample to an equal volume of 2× sample buffer. 10. Load a sample volume of 10 to 15 µl into the bottom of each sample well using a

pipet with a fine tip or equivalent.

Prior to sample loading examine each sample well to ascertain that air bubbles are not trapped in the well.

Electrophorese gel (also see UNIT 6.1)

11. Run samples into the gel matrix at a constant current of 25 mA for ∼30 min or until the sample dye has completely entered the gel.

12. Pause the electrophoresis and decrease the level of the electrophoresis buffer to ~1 mm above the upper surface of the gel. Increase the constant current to 50 mA and run for an additional 3 to 4 hr, or until the marker dye has migrated at least 6 to 7 cm. Blotting the gel

13. Perform immunoblotting to a 0.45-µm Immobilon-P polyvinylidene fluoride (PVDF) membrane in a tank transfer system as described (UNIT 6.2), with the exception of the following conditions:

a. Electrophoretically transfer at a constant current of 100 mA at 4°C overnight. b. Use 0.25× transfer buffer without methanol.

c. Use blocking buffer containing 5% (w/v) nonfat dry milk. Immunodetect protein

14. Perform immunoprobing with directly conjugated secondary antibody as described (UNIT 6.2), except with the following variations which are specific for immunodetec- tion of von Willebrand Factor protein:

a. Dilute primary antibody, rabbit anti-vWF, to a concentration of 1:4000.

b. Dilute secondary antibody, donkey horseradish peroxidase–linked anti-rabbit Ig, to a concentration of 1:2000.

Visualization

15. Visualize von Willebrand factor protein on the PVDF membrane by meticulously following the recommendations enclosed in the ECL Western Blotting Analysis

system. Electrophoresis_and

Visualization with Luminescent Substrates is discussed in UNIT 6.2. To determine the optimal concentration of antibody, run a preliminary gel followed by immunoblotting. Cut the blot into several vertical test strips, each containing 1 to 2 lanes. Use the supplier’s recom- mended concentration of antibody as a starting point and process the test strips with 3- to 10- to 30-fold increased and decreased antibody concentrations in separate containers.

ALTERNATE PROTOCOL

AGARAOSE GEL ELECTROPHORESIS WITH IN-GEL ANTIBODY