Hyaluronic Acid – Size Determination

Gel electrophoresis provides the easiest way to estimate hyaluronic acid (HA) molecular weight from biological samples. However, choosing the correct running conditions is critical for a successful experiment. Here, we provide a general guideline for HA gel electrophoresis with a summary table of HA molecular weight in biological samples to facilitate your HA Gel Electrophoresis experiment.

Sample Preparation

In order to measure the HA molecular weight using gel electrophoresis, HA needs to be isolated from biological samples. The following protocol is based on published literature. Optimization is highly recommended to fit your needs. A summary of HA concentration and MW distribution in biological samples can be found in Table 2 in the full protocol. Spiked biological fluids or solid tissue with monodispersed HA (Echelon Cat. No. HYA-50KEF-1, HYA-500KEF-1, HYA-601KEF-1, HYA-1000KEF-1) can be used to check for potential HA degradation during the isolation process.

Milk (20-30 mL)

1. Heat milk in boiling water for 10 minutes. Cool on ice for 10 minutes.
2. Add Proteinase K to final concentration at 0.5 mg/mL.
3. Incubate at 60 °C overnight. Cool on ice for 10 minutes.
4. Centrifuge at 3,000 x g for 15 minutes at 4 °C.
5. Carefully collect the lower layer into a sterile tube.
6. Repeat Step 4-5 once.
7. Centrifuge at 25,000 x g for 10 minutes at 4 °C. Collect the supernatant.
8. Heat in boiling water for 10 minutes. Cool to room temperature.
9. Repeat Step 7.
10. Dialyze against diH2O overnight using 3.5 kDa cutoff dialysis membrane/cassette.
11. Concentrate sample using centrifugal evaporator.

Serum (0.1 mL)

1. Mix 1-part serum with 3-part of cold ethanol with 1.3% (w/v) sodium acetate.
2. Vortex to mix and incubate on ice for 30 minutes.
3. Centrifuged at 12,000 x g for 20 minutes at 4 °C.
4. Discard supernatant and reconstitute pellet with 0.4 mL diH2O.
5. Repeat Step 1-4 three times.
6. Resuspend the pellet with 0.4 mL 10 mM CaCl2 with 3 mg/mL Proteinase K.
7. Incubate at 50 °C overnight. Inactive Proteinase K by boiling for 15 minutes.
8. Repeat Step 1-4 three times.
9. Resuspend the pellet with diH2O.

Tissue (300-350 mg)

1. Incubate issue in 2 mL 0.15 M Tris, 0.15 M NaCl, 0.01 M CaCl2, and 5 mM deferoxamine mesylate, pH 8.3, containing 40 units
   of Proteinase K at 55 °C overnight.
2. Inactive Proteinase K by boiling for 20 minutes.
3. Centrifuge at 21,000 rpm for 15 minutes at 4 °C.
4. Collect supernatant.

Gel Recipes

These are recommended gel electrophoresis conditions for HA MW Determination based on the expected size of the HA in your sample. Standards are Select-HA Molecular Weight Markers.

Protocol

This protocol is developed as a general guideline for hyaluronic acid (HA) molecular weight determination by gel electrophoresis. We recommend using this guideline as a starting point. Optimize the protocol to fit your needs.

1. Prepare agarose solution with the selected buffer system in an Erlenmeyer flask. Refer to Table 1 for the recommended agarose concentration, gel dimension and buffer system.
2. Melt the agarose using microwave. Do not overheat. Stop heating when the agarose solution is clear. Let the solution warm to touchable temperature.
3. Pour the agarose solution into the gel-casting system of your choice. Insert comb and let the gel set. Once set, cover the gel with the selected buffer. Let gel set overnight at room temperature.
4. If necessary, dilute HA samples with ultrapure water. Mix 15 μL of HA samples or standards with 5 μL 1X Sample Buffer (1:4 dilution). Refer to Table 1 for recommended HA samples and HA standards concentration.
5. Carefully remove comb from gel and place the gel into the electrophoresis apparatus of your choice. Pour the selected buffer to cover the gel.
6. Load 5-20 μL the prepared HA samples or standards (step 4) into the gel. Refer to Table 1 for recommended sample loading concentration & volume.
7. Place electrophoresis unit cover on. Ensure the negative electrode is connected to the top of the gel and the positive electrode to the bottom of the gel.
8. Run gel with decided time and voltage. Refer to Table 1 for recommended running time and voltage.
9. Immediately remove gel after the electrophoresis is completed. Transfer gel into a glass container. Pre-wet the glass container with Stains-All Solution to prevent gel sticking to the glass container.
10. Pour enough Stains-All Solution to cover the gel. Cover the glass container with lid or plastic wrap. Stain gel overnight at room temperature. Protect the glass container from light by wrapping the entire glass container with aluminum foil.
11. Carefully remove the Stains-All Solution. Stains-All Solution can be re-used once.
12. Pour enough Destaining Solution to cover the gel. Cover the glass container with lid or plastic web. Destain the gel at room temperature for minimum overnight. Protect the glass container from light by wrapping the entire glass container with aluminum foil. Change Destaining Solution at least once to facilitate the destaining process.
13. When gel background is reduced, and bands are clear, scan or image gel for record. Gel can be stored in the dark in the Destaining Solution for several days.

Examples

Listed here are examples of the expected HA Concentration and MW Distribution in some types of biological samples. A more comprehensive list can be found in the full protocol. 

References

1. Bhilocha, S.; Amin, R.; Pandya, M.; Yuan, H.; Tank, M.; LoBello, J.; Shytuhina, A.; Wang, W.; Wisniewski, H. G.; de la Motte, C.; Cowman, M. K., Agarose and polyacrylamide gel electrophoresis methods for molecular mass analysis of 5- to 500-kDa hyaluronan. Anal Biochem 2011, 417 (1), 41-9.
2. Cowman, M. K.; Chen, C. C.; Pandya, M.; Yuan, H.; Ramkishun, D.; LoBello, J.; Bhilocha, S.; Russell-Puleri, S.; Skendaj, E.; Mijovic, J.; Jing, W., Improved agarose gel electrophoresis method and molecular mass calculation for high molecular mass hyaluronan. Anal Biochem 2011, 417 (1), 50-6.
3. Huang, X.; Schmidt, T. A.; Shortt, C.; Arora, S.; Asari, A.; Kirsch, T.; Cowman, M. K., A competitive alphascreen assay for detection of hyaluronan. Glycobiology 2018, 28 (3), 137-147.