Method and kit for separating and purifying calprotectin
A calprotectin and kit technology, applied in the field of protein purification, can solve the problems of protein standard and competing samples accuracy impact, damage to protein multimer structure and integrity, cumbersome steps, etc., to save isopoint focusing Steps, easy to promote and apply, the effect of simple steps
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[0049] Secondly, the leukocyte sample is processed by cell disruption to obtain a crude cytoplasmic extract containing the calprotectin. Methods for crushing leukocytes include mechanical crushing, repeated freezing and thawing, and ultrasonic treatment; mechanical crushing using a glass dounce homogenizer is preferred. For example, in the first preferred embodiment, leukocytes are crushed with a homogenizer to obtain a crude cytoplasmic extract, the pH of the cytoplasmic protein extract is 6.5 to 7.0, and the concentration of Tris-HCl in the cytoplasmic protein extract is 15 ~25mmol / L, NaCl concentration is 1.5~2.5mmol / L, MgCl 2 The concentration is 3.0-4.0 mmol / L. In the second preferred embodiment, the preparation method of the crude cytoplasmic protein extract comprises: after the leukocytes are broken in the cell disruption / lysate, centrifuging at a low speed to remove cell debris, and then centrifuging at a high speed to obtain the cytoplasmic protein crude extract. T...
Embodiment 1
[0057] This example provides a method for isolating and purifying calprotectin, the steps are as follows: Step 1. Centrifuge the white blood cells located between red blood cells and plasma from human whole blood, further lyse the red blood cells with a hypotonic solution, and collect the white blood cells by centrifugation. Step 2. Crush white blood cells with a glass cell homogenizer to obtain a crude cytoplasmic protein extract. The components of the cell disruption and cytoplasmic protein extract used are 20mM Tris-HCl, pH6.5-7.0; 2mM NaCl; 3.5mM MgCl 2 . Step 3. Centrifuge in an 850g low-speed centrifuge for 10 minutes, keeping the rotor at 4°C. Step 4. Collect the cytoplasmic supernatant, add EDTA to a final concentration of 5mM, mix well, and centrifuge in a high-speed centrifuge at 15500g for 30 minutes, keeping the rotor temperature at 4°C. Step 5. Collect the centrifuged cytoplasmic supernatant, add ammonium sulfate solution to a final ammonium sulfate concentration...
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