Methods for Immunoblotting of von-Willebrand Factor

 

Immunoblotting of von Willebrand Factor Polymers in Plasma and Platelets using Peroxidase Conjugated Antibody and Chemiluminescence Detection

Principle

Patient platelet-poor-plasma proteins are partially denatured using heat and an anionic detergent, Sodium Dodecyl Sulfate (SDS). This detergent binds to all plasma proteins. The amount of anion bound to the protein is directly proportional to the size of that protein. Thus, when SDS treated plasma proteins are subjected to an electric current they will migrate towards the positive electrode (anode). If forced to move through an agarose “sieving” medium, smaller proteins will migrate faster than larger proteins, and a size dependent distribution of plasma proteins is the result. After electrophoresis, the proteins are transferred out of the gel onto nitrocellulose membrane. The nitrocellulose is then blocked and probed with a peroxidase-conjugated goat anti-vWF antibody. The vWF bands are then visualized using the Amersham Enhanced Chemilumescent Detection System. Normal polymers of von Willebrand factor range in size from 1 to 14 million Daltons in molecular weight. In some variants of von Willebrand’s disease, the larger polymers are missing from the plasma. The gels of higher concentration agarose (3%) are required if triplet substructure is to be defined, while gels of lower agarose concentration (1.5%) allow more straightforward identification of type II variants.

References:

  • Budde, U., Schneppenheim, R., Plendl, H., Dent, J., Ruggeri, Z.M., Zimmerman, and T.S.: Luminographic Detection of von Willebrand Factor Multimers in Agarose Gels and on Nitrocellulose Membranes. Thrombosis and Haemostasis, 63(2): 312-315, 1990.
  • Ruggeri, Z. M., Zimmerman, T.S.: Variant von Willebrand’s Disease. Characterization of two subtypes by analysis of multimeric composition of FVIII/vWF in plasma and platelets. J. Clin. Invest., 65:1318, 1980.
  • Towbin, H., Gordon, J.: Immunoblotting and Dot Immunobinding – Current Status and Outlook. J. Immunol. Meth., 72:313, 1984.

Equipment Required:

  • Power supply, capable of delivering up to 100 V., O.1 A.
  • Horizontal slab gel electrophoresis apparatus (LKB Multiphor 2117).
  • Boiling water bath or Microwave oven.
  • 60oC water bath.
  • Anticondensation lid, Plexiglas, 110 x 250 x 4 mm.
  • Gel pouring kit (gel should be poured using a “sandwich” method).
  • Whatman 3 MM filter paper.
  • Small (1mL) plastic test tubes with caps.
  • Container for washing gel, at least 14 x 28 cm.
  • PVDF membranes (Millipore Corp.) Used as per manufacturers instructions.
  • Transphor Electroblotting Unit (LKB #2005 or Hoefer #TE62X) or equivalent, complete with power supply, cassettes and sponges.

Materials Required:

Description and Source:

  • Antibody: Peroxidase-conjugated goat anti-vWF (ABI #GAVWF-HRP)
  • Tris: Tris(hydroxymethyl)aminomethane. “Trizma Base” Sigma #T-1503
  • Glycine: Sigma #G-7126
  • SDS: Sodium Dodecyl Sulfate: Pierce #28365 (Note: SDS is a respiratory tract irritant. Weigh without raising dust.)
  • BSA: Bovine serum albumin RIA Fraction V: Sigma #A-7888
  • Glacial Acetic Acid: BDH #B10001-78
  • Agarose: HGT(P) Agarose: FMC Corp.
  • ECL Western Blotting Detection System: Amersham Pharmacia #RPN-2106
  • Bromophenol blue: BDH #B20015-20

Preparation:

(Note: use Milli-Q water or comparable purity for all reagents.)

  • Stacking gel buffer: 0.125 M Tris-HCl, pH 6.8
  • To make 500 ml: 7.57 grams Tris to 500 ml water. pH to 6.8 with HCl. (Note : add SDS to 0.1% (w/v) just before use.)
  • Separating gel buffer: 0.375 M Tris-HCl, pH 8.8
  • To make 1 liter: 45.41 grams Tris to 1 liter water. pH to 8.8 with HCl. (Note: add SDS to 0.1% (w/v) just before use.)
  • Tray buffer: 0.05 M Tris, 0.384 M Glycine, 2.5 g SDS (0.1% (w/v)), pH 8.35
  • To make 2.5 liters: 15.0 grams Tris, 72.0 grams Glycine, 2.5 grams SDS, adjust pH to 8.35 if necessary with 1M HCl or 1M NaOH.

Sample prep buffer:

Stock: 0.1 M Tris-HCl, 0.01M EDTA, pH 8.0 Store at 4oC.
To make 100 ml: 1.21 grams Tris, 0.372 g Na2EDTA, pH to 8.0 with HCl.
Working Solution: 0.01 M Tris-HCl, 0.001 M EDTA, 2% (w/v) SDS pH 8.0
For 100 ml: 10 ml stock solution, 90 ml water, add 2.0 grams solid SDS.

  • PBS: 8.0g NaCl and 1.15g Na2HPO4, 0.2g of KH2PO4, and 0.2g of KCl, up to 1 litre, pH to 7.4.
  • PBS-2% (w/v) BSA (Blocker): 10 g BSA to 500 ml PBS, readjust pH to 7.4.
  • Transfer Buffer: To make 6 liters: Add 42.59 g Na2HPO4, 2.4 g SDS, pH to 7.4 with 1 M H3PO4. Store at room temp.
  • Probing Buffer: 5% (w/v) Carnation Skim Milk Powder in PBS + 0.1% (v/v) Tween-20. Adjust pH to 6.5 with 1 M H3PO4. Centrifuge at 3500 X g for 30 minutes just before addition of antibody.
  • Bromphenol Blue: 1% (w/v) Bromphenol blue in water.

Collection and Handling of Specimens:

  • 4.5 ml of venous blood is drawn into a plastic syringe and anticoagulated with 3.8% trisodium citrate (4.5 ml blood to 0.5 ml trisodium citrate). Patient must be fasting.
  • Platelet poor plasma (PPP) is prepared by centrifuging the specimen in table-top centrifuge at 3000 rpm (100 x g) for 10 minutes at room temperature.
  • PPP is carefully removed with a plastic transfer pipette, placed in a plastic test tube and frozen and stored at -20oC or below.

Sample preparation for platelet vWF multimeric analysis:

  1. Collect blood as above, but spin for 5 min @ 180 X G
  2. Perform platelet count on the PRP and calculate volume required to give total of 2.5×108 platelets.
  3. Spin required volume of PRP in a microfuge for 1 minute to achieve pellet of 2.5×108 platelets.
  4. Save supernatant and freeze (for plasma vWF analysis) and freeze platelet pellet
  5. Thaw pellet and add 900 ul H20.
  6. Freeze & thaw 5 times (to lyse platelets).
  7. Spin in microfuge, 1 min & remove supernatant (contains released vWF)
  8. Add equal volume of 0.5% Triton X-100 (v/v) in 5% SDS (w/v) and incubate for 15 minutes at 60oC.
  9. Spin in microfuge and apply to gel as for plasma. (Note: Can use 1×108 platelets but scale everything down.)

Method

A. Pouring the gel (“sandwich” method):

  1. The sandwich or mold consists of two glass plates, a U-shaped spacer, 1 mm thick and four clamps.
  2. Place 1 mm spacer between the two glass plates. Clamp assembly together and place mold(s) in 60oC oven for 15 minutes.