Process for separating protein of Chinese medicine
A protein and traditional Chinese medicine technology, applied in the field of separating traditional Chinese medicine proteins, can solve the problems of loss of activity, impurity of the separated sample, and complicated separation work.
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Embodiment 1
[0040] Preparation of Magnetic Nanoparticles
[0041] Prepare magnetic nanoparticles according to the method described in Chinese patent application 03142274.8, the specific method is as follows:
[0042] (a) Preparation method of the inner core layer of magnetic nanoparticles
[0043] The core layer of magnetic particles was prepared by improved chemical co-precipitation, the specific method is as follows: prepare FeSO 4 ·7H 2 O and FeCl 3 ·6H 2 O mixed solution and NaOH solution. In a mixed solution of iron salts, Fe 2+ The ion concentration is 0.1~0.2mol / l, Fe 3+ The concentration of ions is 0.1-0.3 mol / l, and the concentration of NaOH solution is 2-3 mol / l. Under vigorous stirring, the NaOH solution whose volume is half of the volume of the mixed salt solution was slowly added dropwise into the mixed salt solution. Aging the obtained solid precipitate at 40°C to 60°C for 12 hours, washing the precipitate several times with double distilled water, filtering and dr...
Embodiment 2
[0047] Preparation of magnetic nanoparticles modified by amino groups
[0048]Get 15mg of the magnetic nanoparticles coated with silicon dioxide prepared in Example 1, add it to the mixed solution (1:1, v / v) of methanol and glycerol in 2ml milliliters, sonicate for 30 minutes, and then take 0.01 g of AEAPS[N-(2-aminoethyl)-3-aminopropyltrimethoxysilane] was added to the mixture, and ultrasonically treated for 10-60 minutes to mix the solution evenly. Under the reaction conditions of 15-90°C React for 2-3 hours, then take out the particles, wash them with methanol and water for 2-3 times, dry them in vacuum at 150°C for 2 hours, and collect the particles, which are amino-modified magnetic nanoparticles, referred to as MNP-NH 2 .
[0049] Using the prepared amino-modified magnetic nanoparticles, prepare PBS solutions containing 20 μg / ml, 40 μg / ml, 60 μg / ml and 80 μg / ml MNP. Take 20 microliters of the above solutions and mix them with 200 microliters of BSA solution (1 mg / ml), ...
Embodiment 3
[0052] Handling of Biological Samples
[0053] Use different methods for different biological samples;
[0054] (a) Isolation of proteins from hemogenous cells:
[0055] Cells were lysed using conventional lysis methods. The lysate contains 0.5% NP40; 10mmol / L Tris·Cl (pH8.0); 10mmol / L NaCl; 3mmol / L MgCl 2 ; 1 mmol / L PMSF (phenylmethylsulfonyl fluoride). This lysate is suitable for the isolation of proteins in hemogenous cells and the study of signal transduction.
[0056] (b) Isolation of soluble proteins from botanicals:
[0057] According to the traditional folk medicine method, first decoct various Chinese medicinal plants (see Table 1) for 20 minutes with a slow fire, collect the decoction, centrifuge at 3000r / min at room temperature for 15 minutes, and take the supernatant directly for protein separation.
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