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The purpose of hemoglobin electrophoresis is to detect and confirm various normal and abnormal hemoglobin.
According to different hemoglobin with different charges, the isoelectric point is different. In a certain pH buffer, the isoelectric point of hemoglobin is negatively charged when it is lower than the pH of the buffer, and the electrophoresis moves toward the anode in the electric field. Hb carries a positive charge to the cathode. Under a certain voltage, after a certain period of electrophoresis, different hemoglobins have different charges and different molecular weights. The direction and speed of the hemoglobin are different, and the respective bands can be separated, and the electrophoresis bands can be colorimetrically or electrophoresed. Scanning allows quantitative analysis of various hemoglobins. The most commonly used is the pH 8.6 acetate membrane electrophoresis.
The glycol group (CHOH-CHOH) in the cytoplasm containing glycogen or polysaccharides (such as mucopolysaccharides, mucins, glycoproteins, glycolipids, etc.) is oxidized by periodic acid to be converted into dialdehyde groups. (CHO-CHO), combined with colorless magenta in Schiff's reagent, forms a purple-red dye and deposits in the intracellular polysaccharide. This reaction is known as a periodic acid-Schiff (PAS) positive reaction, formerly known as glycogen staining.
experimental method
material:
Buffer
(1) pH 8.6 TEB buffer: Weigh 10.29 g of Tris, 0.6 g of EDTA, 3.2 g of boric acid, and add distilled water to 1000 ml.
(2) Borate buffer: Weigh 6.87 g of borax, 5.56 g of boric acid, and add distilled water to 1000 ml.
2. Acetate film, electrophoresis instrument, sampler, spectrophotometer, cuvette.
method:
1. Preparation of hemoglobin solution
Take heparin or sodium citrate anticoagulation 3 ml, centrifuge at 2000 rpm for 10 minutes, discard the plasma; wash the red blood cells three times with physiological saline (750 rpm, centrifugation for 5 minutes), centrifuge at 2200 rpm for 10 minutes, discard the supernatant Add an equal amount of distilled water; add 0.5 times volume of carbon tetrachloride, shake vigorously for 5 minutes, centrifuge at 2200 rpm for 10 minutes, and then suck the upper layer of Hb solution for later use.
2. Dipping film
The cellulose acetate film was cut into 3 cm × 8 cm paper strips, immersed in pH 8.6 TEB buffer, soaked, taken out, and blotted dry with filter paper.
3. Spotting
10 μl of the hemoglobin solution was taken with a pipette, and then vertically sampled onto the cellulose acetate membrane (matte surface) at a distance of 1.5 cm from the edge.
4. Electrophoresis
Pour the borate buffer as an electrophoresis buffer into the electrophoresis tank, place the spotted cellulose acetate membrane on the electrophoresis tank holder, and place it on the cathode end at 200 V for 30 minutes.
5. elution
Cut HbA and HbA2 separately, put them into a test tube, add 15 ml of distilled water and 3 ml respectively, gently shake them, and mix them after the hemoglobin is completely eluted.
6. Colorimetric
The eluate was zeroed with distilled water and the absorbance was measured at 415 nm.
7. Calculation
HbA2 (%) = HbA2 tube absorbance / (HbA tube absorbance × 5 + HbA2 tube absorbance) × 100%
Experimental result calculation
pH8.6 TEB buffer acetate fiber electrophoresis reference range: HbA>95%, HbA2 1%-3.1%
Precautions
1. The electrophoresis time should not be too long. When the electrophoresis, the vinegar membrane cannot be dried. Therefore, it should be observed that the HbA and HbA2 are separated and the electrophoresis is stopped. The electrophoresis time is too long and the zone is diffused and blurred.
2. The amount of spotting should not be too much, such as too much hemoglobin solution, the ribbon is easy to fall off or the dye is not transparent, and the false positive result of relative increase of HbA may occur;
3. Avoid contamination of the cellulose acetate membrane with protein;
4. The current should not be too large, otherwise hemoglobin can not be separated;
5. A standard control of the normal person and the necessary known abnormal hemoglobin should be performed at the same time.
Experimental principle