ACID HEMOGLOBIN(E) K20 QUICK REFERENCE GUIDE

K2003
ACID HEMOGLOBIN(E) K20 QUICK REFERENCE GUIDE
Reagent Preparation
• Citrate Buffer- Each vial of stock buffer solution to be diluted up to 1L with DI H2O (stable
for 1 year).
• Amido Black Stain- Each vial of stock acid violet stain to be diluted to 300ml with DI H20
(stable for 1 year).
• Destain Solution- Dilute destain solution -1ml stock to 1L with DI H20 (Stable for 1 week).
• Hemolyzing Solution- Ready to use. ( Stable until expiration date on vial).
Sample preparation
• Use fresh anticoagulated blood for analysis.
• Centrifuge anticoagulated sample at 5,000 rpm for 5 minutes.
• Wash the red blood cells 2 times with 10 volumes of saline.
• Hemolyze 10µL of packed red cells with 130uL of Hemolysis reagent.
• Vortex for 10 seconds and incubate 5 minutes at room temperature
• Dilute the hemolyzate 4 times with saline (example 25ul hemolyzate +75 ul saline).
Procedure
1. Apply 10µL of hemolyzed sample into the applicator wells.
2. Place applicator(s) in wet storage chamber (teeth up) for ≥ 5 min. (max 8 hr. at 2 - 8 °C)
3. Place the HYDRAGEL K20 applicator carrier on a flat surface and raise the part of the
applicator carrier with the numbered notches.
4. Pipette 120µL DI water on the lower third of the frame printed on the HYDRAGEL K20 carrier
with the numbered notches.
5. Blot gel quickly using thin blotter paper.
6. Place the gel plate (the gel side up) with its edge against the stop at the bottom of the printed
frame. Roll gel down. Avoid bubbles.
7. Lower the applicator carrier with the numbered notches down to the intermediate position with
the switch in high position, i.e. turn switch completely away from operator, until click is heard.
8. Place applicator comb into position 9 of the carrier.
9. Lower the carrier so that the applicator contacts the gel surface. DO NOT PRESS DOWN ON
CARRIER
10. After 1 minute, turn the switch to rise up the applicator, remove the applicator and discard.
11. Prepare buffer and electrophoresis chamber appropriately.
12. Place gel on the chamber bridge with the gel side facing down; the gel should dip about 1cm
into the buffer on each side.
13. Plug the chamber into the power supply.
Sebia recommends using the constant voltage mode. Utilize these migration conditions for
optimum performance. Set all other parameters to maximum accepted values.
Migration Conditions SEBIA K20 Maximum Values
Volume of buffer per compartment 150ml I = 200mA
Total buffer volume 300ml P = 60 W
Migration time 35 minutes VH = 9999
Constant voltage
40 V
After migration, unplug the chamber and carefully remove the gel plate.

14. Hot air Fixation- Dry the gel completely in the incubator-dryer at 80 C.
Fixation with fixative solution
• Place gel into gel holder.
• Fill one tank with 150ml of fixative solution. Note: fixative should be prepared at
lease 15 minutes prior to testing, by combining (vol./vol.)60% ethanol,10% acetic
acid and 30% DI water.
• Immerse the gel in the fixative for 15 minutes.
• Remove the gel and dry it with 80 C airflow. Note: Gel must be completely dry.
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15. Immerse dried and cooled gel vertically in staining solution for 5 minutes.
16. Destain in three successive baths of destaining solution until the background is completely
colorless and clear.
17. Soak up excess liquid on the gel with a tissue paper and dry gel in 80 C air stream.
See package insert for procedural details and further information.
Rev 01.15.03