Von Willebrand Factor Multimer Analysis by Low Resolution SDS-Agarose Gel Electrophoresis

Materials and Reagents

Material

1. Flatbed electrophoresis apparatus and Multiphor II electrophoresis unit (with ceramic cooling plate), GE Healthcare, IL, USA (or equivalent)

2. Power Supply

3. Cryo thermostat, Modell MultiTemp III, (Cytiva, Uppsala, Sweden)

4. Hairdryer

5. Horizontal shaker

6. pH-meter

7. Vortex mixer

8. Water bath (up to 90 °C)

9. Pipettes and tips

10. Plastic test tubes 12 × 55 mm

11. Glass and plastic laboratory trays, approximately 14 × 26 × 6 cm

12. Glass plates, float glass, 11.5 × 23.0 × 0.2 cm (or equivalent)

13. Clamps for electrophoresis plates

14. Rubber seals for gel plates, U-shaped cut, approximately 1.5 mm thick

15. Rubber seals rectangular cut, 10 mm long, 4 mm wide, 1.5 mm thick

16. Disposable syringes, cannulas, and locking cone

17. GelbondFilm for agarose gel 124 × 258 mm (product: 80112932; Cytiva, Uppsala, Sweden)

18. Chromatography paper 1CHR (Whatman WHA1001917; Sigma, Vienna, Austria) and 3CHR (Whatman WHA3003917; Sigma, Vienna, Austria)

19. Paper towels/kitchen roll

20. Laboratory balances

    
    

Reagents

1. Purified water

2. Agarose SEAKEM HGT (prod. 50040; Lonza, Rockland, USA)

3. Agarose Biozym Plaque Agarose (prod. 840101; Biozym Scientific GmbH, Oldendorf, Germany)

4. Tris(hydroxymethyl-aminomethane)

5. Sodium dodecyl sulfate pellets (SDS)

6. Ethylen-diamin-tetraacetic acid-di-sodium salt EDTA

7. NaCl

8. Glycine

9. Bromophenol-blue

10. Triton X100

11. Na₂HPO₄·2H₂O

12. NaH₂PO₄·H₂O

13. Antibody rabbit anti-human-VWF (product A0082 DAKO, Agilent Technologies, Glostrup, Denmark)

14. Secondary antibody goat-anti-rabbit-IgG-ALP-conjugated. (product 111-055-003; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA)

15. AP Conjugate Substrate Kit (product 1706432, BioRad Laboratories, Hercules, CA, USA)

16. Human normal plasma pool (e.g., Precision Biologics, Rockville, USA)

17. 0.9% NaCl

18. Separation gel buffer (see Recipes)

19. Stacking gel buffer (see Recipes)

20. Sample buffer (see Recipes)

21. Running buffer for electrophoresis apparatus (see Recipes)

22. Bromophenol blue solution (see Recipes)

23. PBS-Buffer for immuno-staining (in gel staining) (see Recipes)

24. Washing buffer/antibody dilution buffer (see Recipes)

Equipment

1. Densitometer GS-900 (BioRad Laboratories, Hercules, CA, USA)

2. Software Image Lab (BioRad Laboratories, Hercules, CA, USA) and/or Image Quant TL (Cytiva, Uppsala, Sweden)

3. Multiphor II Electrophoresis-System with cooling unit (Figure 1).

4. Microwave oven

   
   
   Figure 1. Electrophoresis system: Cooling unit (left) and flatbed apparatus (right)

Procedure

1. Prepare the glass cassette for hand-made agarose gel

   1. On a plane glass plate, glue rectangular rubber seals (10 mm long; 4 mm wide; 1.5 mm thick) on the long side of the glass plate (1.5 cm under the longitudinal edge)—14 pieces at regularly spaced distance. These will form the loading wells for samples (Figure 2A).

   2. Place a separate plane glass plate on the bench (11.5 × 23.0 × 0.2 cm). Cut gelbond film to a length of 23 cm. With a few drops of purified water, fix the gelbond film with its hydrophobic side on the glass plate, avoiding air bubbles (Figure 2B).

   3. Wet the U-shaped rubber seal and place it on the gelbond film (Figure 2C). Place the other glass plate with the loading rubber seals on top, so it forms a casting mold (cassette) (Figure 2D).

   4. Fix the glass cassette with clamps (Figure 2E).

      
      

      
      Figure 2. Preparation of the gel cassette.

      
      

2. Casting of separation gel

   Caution: Hot liquids, handle with care. Wear protective equipment to prevent injury.

   1. Prepare a 1% agarose gel. Suspend 0.5 g agarose SEAKEM HGT with 50 mL of separation gel buffer and heat the solution in a microwave oven (e.g., 900 W; interrupt heating when liquid starts to foam, remove from oven, and gently agitate; repeat procedure until complete dissolution). Draw this solution into a 50 mL syringe, close the syringe with a cone, and incubate it in a water bath at approximately 90 °C for approximately 30 min.

   2. Place the glass cassette upright in a pre-warmed water bath at 60 ± 5 °C. To cast the gel, place a cannula on the syringe and slowly fill the hot agarose into the glass cassette (on one upper side of the glass cassette) until approximately 1 cm below the loading wells.

   3. Take the glass cassette out of the water bath and allow the gel to set. Keep at room temperature (RT) for approximately 1 h, standing upright.

      
      

3. Casting of stacking gel

   Caution: Hot liquids, handle with care. Wear protective equipment to prevent injury.

   1. Suspend 0.2 g agarose SEAKEM HGT with 25 mL of stacking gel buffer in the microwave oven until complete dissolution. Cool to approximately 60–70 °C, and pipette the agarose on top of the separation gel until reaching the upper end of the cassette.

   2. Once the cassette is fully filled, keep a small volume of stacking gel for Procedure F.

   3. Allow the gel to set for approximately 1 h at RT. Open the glass cassette by carefully removing the clamps and the upper glass plate. Take out the gelbond film with the adhesive agarose gel.

      
      

4. Preparing the electrophoresis apparatus

   1. Turn on the cryostat to cool down the flatbed ceramic plate at approximately 10 °C.

   2. Add the running buffer (approximately 1 L per flatbed chamber) to the reservoirs at each end of the gel chamber.

   3. Pipette a few drops of running buffer on the ceramic plate, and place on top the gelbond film with the gel, with the loading wells on the cathodic side.

   4. Filter papers (1CHR) soaked with running buffer are placed as follows: five layers of paper (23 cm × 15 cm), one end in the cathodic buffer vessel, the other end attached to the cathodic end of the gel (do not overlap the gel), and on top another five layers slightly overlapping the agarose gel (keep at approximately 1 cm distance to the sample application wells). Repeat the same procedure on the anodic end of the gel, with five layers of 23 cm × 15 cm paper and five layers of 23 cm × 23 cm paper.

      
      

5. Sample preparation/loading

   Caution: Hot liquids, handle with care. Wear protective equipment to prevent injury.

   1. Dilute samples and assay control to the same VWF concentration (preferably 1 IU VWF:Ag/mL), using 0.9% NaCl as diluent.

   2. Mix 0.2 g Biozym Plaque Agarose with 10 mL of sample buffer, and heat in the microwave oven until completely dissolved. Cool down in water bath to approximately 60 °C.

   3. Mix 50 µL of diluted sample, 50 µL of sample buffer, 10 µL of bromophenol blue solution, and 100 µL of Biozym Plaque Agarose.

   4. Incubate at 60 ± 5 °C for 20 ± 5 min.

   5. Load a volume of 50 µL per loading well.

      
      

6. Electrophoresis

   1. Close the lid and start the electrophoresis.

   2. Running time: 15–20 h.

   3. Running conditions: 25 mA constant for 1–3 h, until samples are fully run out of the wells. Interrupt the run to fill the empty wells with stacking gel (30 µL). Continue the separation with 8–10 mA for 14–16 h, until the bromophenol blue line has reached the end of the gel.

      
      

   After separation, VWF multimers are visualized by either direct in-gel staining or after blot transfer. The protocol below describes the in-gel procedure only.

   
   

7. In gel immuno-staining

   Drying the gel

   1. After finishing the gel run, remove the filter papers and wash the gel with purified water on a horizontal shaker for approximately 1–3 h, changing the water for a minimum of five times. The bromophenol blue line should no longer be visible.

   2. Press the gel for 45 min: Place two chromatography paper sheets (3CHR) soaked with purified water on the gel, two more dry papers, and five more kitchen towels. Place one glass plate on top, and press with approximately 2 kg (e.g., place two 1 L bottles with water on top).

      After pressing, remove all paper sheets, fix the gel on the bench, and carefully dry the gel with a hair dryer for approximately 30 min.

   Immuno-staining

   Note: The following procedure is for detection of human VWF. For visualization of VWF from other species, appropriate antibodies need to be selected.

   
   

   Primary antibody: rabbit anti-human-VWF (A0082)

   1. Place the gel in a tray.

   2. The antibody is diluted to 1 µg/mL in washing buffer/antibody dilution buffer. Prepare a solution of 100 mL per gel, and add to the tray.

   3. Incubate the gel under gentle agitation (shaker) for approximately 16 h.

   4. Pour off the antibody solution and wash the gel with washing buffer/antibody dilution buffer for 5 h.

      
      

   Secondary antibody: goat-anti-rabbit-IgG-ALP-conjugated

   5. Dilute the secondary antibody with washing buffer/antibody dilution buffer to 0.6 µg/mL (100 mL per gel).

   6. Place the gel into the diluted secondary antibody and incubate under gentle agitation for approximately 16 h.

   7. Pour off the secondary antibody and wash the gel for 1 h in washing buffer/antibody dilution buffer.

   8. Repeat the washing procedure for a further 3–4 h in PBS buffer for immunostaining (without Triton X100), by changing the buffer a minimum of five times.

      
      

   Immuno-staining according to package insert from AP-Kit (Bio-Rad)

   9. Prepare a solution with 4 mL of kit buffer, 1 mL of reagent A, and 1 mL of reagent B from the AP-Kit. Fill up to 100 mL with purified water.

   10. Use a fresh tray. Place the gel in the staining solution and incubate on a shaker at RT for approximately 10–30 min. The incubation time depends on the band intensity. The bands must be clearly visible, without significant background stain.

   11. Place the gel in purified water to stop staining. Wash with purified water for approximately 20 min.

   12. Fix the gel horizontally on the sides with adhesive strips, and dry the gel either by air drying or with a hair dryer.

Data analysis

Gels can be analyzed visually through side-by-side analysis—the number of multimers, countable with the naked eye, can be recorded (Figure 3) or by densitometry (Figure 4).

Figure 3. VWF multimer gel. Multimer gel. Lanes 1–4: recombinant VWF. Lane 5: normal human plasma pool.

For densitometric analysis, the gels are scanned with a densitometer (GS 900) and analyzed with an appropriate software (for example, Image Lab or Image Quant TL).

For each lane, the distance between the top of the separation gel and the lowest multimer band (corresponding to VWF dimers) is assigned a migration value of 1.0. The distance of the largest VWF multimer band in the sample lane is then measured and expressed as a fraction of this, denoted relative migration distance (R_f). The proportion of the total migration distance of the VWF dimer band that is occupied by all of the other multimers in the sample lane is therefore 1-R_f (Figure 4).

Figure 4. Densitometric analysis (details are described in Turecek et al., 2021). VWF: Ag concentration of the test sample was adjusted to 1 IU/ mL and separated on a 1% agarose gel, followed by immunostaining and densitometry. Multimer pattern (left) and densitogram (right). “a” is the migration distance of the largest VWF multimer, “b” is the migration distance of the smallest VWF (dimer), assigned a migration value of 1.000.

To enable a comparison between different electrophoresis gel runs, this value is reported as a percentage of the 1-R_f value for a normal plasma sample separated on the same gel. An increased percentage value indicates the presence of UHMW VWF multimers in the test sample.

In this example, the relative migration distance (Rf) of the largest VWF multimer was 0.392. The proportion of the separation lane that is occupied by VWF multimers (smallest to largest) is therefore 1-Rf, in this example, calculated as 1.000 – 0.392 = 0.608. The 1-Rf value for the test sample may be calculated as a percentage of the 1-Rf for a control sample run on the same gel to normalize results.

Recipes

1. Separation gel buffer

   pH	pH 8.8 ± 0.1
   Ingredients	0.375 M Tris
   	0.1% SDS
   Preparation: Dissolve 45.43 g Tris and 1 g SDS with approximately 800 mL of purified water, adjust to pH 8.8 ± 0.1 with 25% HCl and fill up to 1,000 mL.

   
   

2. Stacking gel buffer

   pH	pH 6.8 ± 0.1
   Ingredients	0.125 M Tris
   	0.1% SDS
   Preparation: Dissolve 7.57 g Tris and 0.5 g SDS with approximately 400 mL of purified water, adjust to pH 6.8 ± 0.1 with 25% HCl and fill up to 500 mL.

   
   

3. Sample buffer

   pH	pH 8.0 ± 0.1
   Ingredients	0.01 M Tris
   	0.001 M EDTA
   	2% SDS
   Preparation: Dissolve 0.121 g Tris, 0.0372 g EDTA, and 2.0 g SDS with approximately 80 mL of purified water, adjust to pH 8.0 ± 0.1 with 25% HCl and fill up to 100 mL.

   
   

4. Running buffer for electrophoresis apparatus

   pH	pH 8.4 ± 0.1
   Ingredients	0.05 M Tris
   	0.384 M Glycine
   	0.1% SDS
   Preparation: Dissolve 12.11 g Tris, 57.65 g Glycine, and 2.0 g SDS with 2,000 mL of purified water. The pH value automatically reaches 8.4 ± 0.1.

   
   

5. Bromophenol blue solution

   Ingredients	Bromophenol blue
   	Sample buffer
   Preparation: Dissolve 0.1 g Bromophenol blue in 50 mL of sample buffer.

   
   

6. Washing buffer/antibody dilution buffer

   Ingredients	Triton X100
   	PBS-buffer for immuno-staining
   Preparation: Dilute 10 g Triton X100 in 1,000 mL of PBS buffer for immuno-staining.

   
   

7. PBS-Buffer for immuno-staining (in gel staining)
   

   pH	pH 7.4 ± 0.1
   Ingredients	0.01 M Na2HPO4·2H2O
   	0.01 M NaH2PO4·H2O
   	0.154 M NaCl
   Preparation: Mix the two buffer solutions to reach pH 7.4 ± 0.1. Preparation of PBS-solution secondary Phosphate: Dissolve 17.8 g Na2HPO4·2H2O and 90 g NaCl in 10 L of purified water. Preparation of PBS-solution primary Phosphate: Dissolve 5.52 g NaH2PO4·H2O and 36 g NaCl in 4 L of purified water. Dilute approximately 5 L of PBS-solution secondary Phosphate with PBS-solution primary Phosphate to reach pH 7.4 ± 0.1.

   
   

8. Washing buffer/antibody dilution buffer 
   

   Ingredients	Triton X100
   	PBS-buffer for immuno-staining
   Preparation: Dilute 10 g Triton X100 in 1,000 mL of PBS buffer for immuno-staining.

Acknowledgments

The basic protocol has been implemented in our laboratory in the mid 1980s by Fritz Elsinger. Since then, we are permanently pursuing VWF multimer analysis using the described electrophoresis method. Over the years the protocol was consistently optimized by subtle improvements. These had been elaborated in collaboration with many technicians working on the method. We acknowledge expert technical support from Alena Kratochwil, Ingrid Neunteufl, Gertrude Maurer, Gabriele Müllner, Nina Pruckner, Astrid Bogat, and Julia Lasnik.

Competing interests

HG, PLT and MS are employees of Baxalta Innovations GmbH, a Takeda company, PLT holds relevant Takeda patents and Takeda stock.

References

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