Separation and purification of serum albumin
Purpose requirement
Understand the principle of ion exchange chromatography by separating and purifying serum albumin
Experimental principle
The cation exchangers commonly used in ion exchange chromatography have weakly acidic carboxymethyl cellulose, and the anion exchanger has weakly basic diethylaminoethyl cellulose.
The mixture of proteins binds to the acidic gene or basic group of the cellulose ion exchanger, and the magnitude of the binding force depends on the electrostatic attraction of the oppositely charged groups, which in turn is related to the pH of the solution, because the ion exchanger and protein are determined. The degree of dissociation. The presence of a salt can reduce the electrostatic attraction between the dissociation group of the ion exchanger and the opposite charge boundary of the protein. Therefore, elution of the adsorbed protein is achieved by changing the pH or ionic strength. The protein with low binding force to the ion exchanger is first eluted from the column. In this experiment, salt albumin and ion exchange chromatography were used to separate and purify serum albumin. First, salting out is used for preliminary separation. In the semi-saturated ammonium sulfate solution, serum globulin is precipitated, and after centrifugation, the supernatant contains albumin. The second step is desalting by gel chromatography. The relative molecular mass of the protein is much larger than that of ammonium sulfate. The appropriate gel fractionation range is selected, and the salt in the crude separation sample is removed according to the molecular sieve effect. Finally, the target protein is purified by ion exchange chromatography, and the pl of the protein is used to select a suitable pH buffer solution to change the ionic strength of the solution to separate the target protein impurities. The desalted sample is dissolved in 0.025 mol/L acetic acid buffer solution. Add to the diethylaminoethyl cellulose column. At this pH, DEAE cellulose has a positive charge and can adsorb negatively charged albumin. The y-globulin is positively charged and does not be adsorbed, so it flows out directly. At this time, the obtained y-globulin is purified. By increasing the salt concentration, the B-globulin and part of the a-globulin on the ion exchange column can be eluted. The salt concentration is then increased to 0.3 mol/L ammonium acetate, and the albumin is eluted, at which point the pure albumin is collected.
Reagents and equipment
1, reagent
(2), 0.3 mol/L, NH4Ac buffer (pH 6.5). Weigh 23.13g of NH4Ac, add 800mL of distilled water to dissolve, adjust pH 6.5 with dilute ammonia or dilute HAc, and add distilled water to 1000mL.
(2) 0.06 mol/L NH4Ac buffer (pH 6.5). 0.3 mol/L NH4Ac was diluted with 5 as distilled water.
(3), 0.02 mol/L NH4Ac buffer (pH 6.5). 0.06 mol/L NH4Ac was diluted with distilled water for 3 times.
The above three buffers must be prepared. After dilution with distilled water, the pH should be tested with a pH meter. Since NH4Ac is a volatile salt, the solution must be stored in a sealed state to prevent changes in pH and concentration, otherwise the purity of the isolated protein will be affected.
(4), 1.5 mol/L, NaCI-0.3 mol/L, NH4Ac. 87.7 g of NaCI was weighed and dissolved in 0.3 mol/L, NH4Ac (pH 6.5) to 1000 mL.
(5), saturated ammonium sulfate solution. Weighing (NH4)2SO4, 850g is added to 1000mL of distilled water, stirred and stirred in a water bath of 70-80 °C, and left at room temperature overnight. The bottle is low-precipitated with white crystals, and the supernatant is a saturated ammonium sulfate solution.
(6) 200 g/L sulfosalicylic acid.
(7), 10g/L, BaCI2
2, materials
Serum (human and other animals)
3, equipment
Column (1 cm × 15 cm, 1 cm × 25 cm), holder, recessed reaction plate, dropper, water bath.
Method of operation
1. Preparation of the column
(1), Sephadex G-25 column
a. Preparation of the gel: Weigh the glucan gel G-25 dry glue (25 g of gel residue on a 100 mL gel bed), add about 50 mL of distilled water per gram of dry glue, shake gently, and place in a boiling water bath. In 1h, and often shake to make the bubbles escape, take out and cool. After the gel was precipitated, the supernatant was decanted, and 2 volumes of 0.02 mol/L, NH4Ac buffer (pH 6.5) was added and mixed. After standing for a while, the gel particles were allowed to settle, and the supernatant was decanted. The treatment was repeated once more with 0.02 mol/L, NH4Ac buffer (pH 6.5).
b. Loading: The column is vertically fixed on the rack, a small amount of 0.02mol/L, NH4Ac buffer solution is added into the tube, and the treated gel suspension is continuously injected into the chromatography tube until the desired gel bed height is reached. (20cm). When packing the column, it should be noted that the gel particles are uniform, the bed surface should be flat, and there should be no interface or air bubbles in the gel column bed. After loading, the column was connected to a constant pressure liquid storage bottle, the flow rate was adjusted to 2 mL/min, and the equilibrium was washed with 0.02 mol/L, NH4Ac buffer (pH 6.5).
c. Regeneration and preservation: This gel column can be used repeatedly. After each use, it can be reused after washing the balance with the required buffer.
In order to prevent gelatinization, it should be washed with 0.2g/L and NaN3 buffer after use. If it is not used, the gel particles should be poured out from the column, add 0.2g/L, and keep NaN3 wet at 4°C. refrigerator.
(2), DEAE-cellulose chromatography column
a. Acid-base treatment: Weigh DEAE-cellulose dry powder, weigh 1:10-15, dry with 0.5mol/L, NaOH for 30min, wash to pH7.0, use 0.5mol/L, HCI soak for 30min Washed to pH 4.0, soaked in 0.5mol / L NaOH for 30min, then washed to pH7.0
b. Loading and balance: The acid-base treated DEAE-cellulose was immersed in 0.02 mol/L, NH4Ac buffer solution with dilute acetic acid to adjust the pH of the suspension to 6.5, the resin was allowed to sink, and the supernatant was removed. According to this method, it was washed three times with 0.02mol/L, NH4Ac buffer, and each time it was washed, it was heavier than pH until the pH was 6.5 and remained unchanged.
c. Regeneration and preservation: This gel column can be used repeatedly. After each use, 1.5 mol/L, NaCI-0.3 mol/L, NH4AC buffer can be used for washing, and then washed with 0.02 mol/L, NH4Ac buffer (pH 6.5), and then reused. After repeated use, if there are more impurities or the flow rate is too slow, the cellulose can be poured out, firstly washed with 1.5~2mol/L, NaCI, washed with water, and then repacked with acid and alkali as described above. If not used, it should be stored in a buffer containing 1% n-butanol in a wet state to prevent mildew.
2, separation and purification
(1), salting out
Take 0.5mL serum, slowly add 0.5mL saturated ammonium sulfate while shaking, mix and let stand for 10min at room temperature, then centrifuge 3000r/min, 10min, carefully aspirate the supernatant with a dropper for purification of albumin. .
(2), desalting
a. Loading: Carefully control the buffer surface on the column just after washing with 0.02 mol/L, NH4Ac buffer (pH 6.5) to just drop to the surface of the gel bed. The crude protein solution obtained by salting out was immediately and carefully applied to the surface of the bed with an elongated dropper to bring the sample level down to the surface of the bed. The column wall was washed with 2 mL, 0.02 mol/L, NH4Ac buffer, placed in a gel bed, and washed three times, and connected to a constant pressure reservoir.
b. Collection: continue to wash with 0.02mol/L, NH4Ac buffer, contact the sulfosalicylic acid solution in the concave hole of the reaction plate, check whether the effluent contains protein at any time, and if it flows out into the concave hole, it will contact the sulfonate. When the salicylic acid solution is used, white turbidity or precipitation appears, indicating that the protein has flowed out. Immediately collect the effluent protein liquid. When the albumin is desalted, 3 to 4 tubes can be collected continuously, and about 1 mL is collected from each tube. Two drops of the effluent from each tube are collected in each well of the reaction plate, and 1 drop of 10 g/L is added, and BaCI2 is checked. Whether there is any, the tubes that are not merged, some are discarded
c. Balance: The gel column after protein collection continues to be washed with 0.02mol/L, NH4Ac buffer, and the effluent is checked with 10g/L, BaCI2. After the test is negative, continue to wash 1~2 bed beds. volume. The gel column is regenerated and can be reused.
(3), purification
After the desalted albumin sample was applied to the column, it was washed with 0.06 mol/L, NH4Ac buffer (pH 6.5), and after about 30 mL, the buffer surface on the column was lowered to be flush with the cellulose bed surface. Then, it was replaced with 0.3 mol/L, NH4Ac buffer (pH 6.5), and 20% sulfosalicylic acid was used to check whether the effluent contained protein. When there is protein outflow, immediately collect 3 tubes, 10 drops per tube, which is the purified albumin solution, and take two tubes with high protein concentration for content determination and purity identification.
Precautions
(1), accurately prepare NH4Ac buffer and strictly adjust its pH to 6.5.
(2) Keep the bed surface intact. Do not rush the surface of the column bed when adding or adding buffer.
(3) Pay attention to collect samples when washing, do not run off the sample, and pay attention to the column not to drain and enter the bubble.
HQD Cuvie Plus is a revolutionary 1200 Puff disposable pod device designed to be a rewarding alternative to cigarettes.
This compact AIO disposable device includes an 850mAh battery module and a pre-filled e-liquid pod containing 50mg of salt nicotine. The resolute aluminum exterior allows for a reliable device that is ergonomic due to its discreet, cylindrical design. The 850mAh inbuilt battery means that this is a long-lasting device, the level of charge is indicated through the LED light at the base of the device. With an inhale activation, there is no maintenance required just simply inhale straight away to begin vaping. What makes HQD Cuvie Plus so impressive is the number of puffs and e-juice capacity. The capacity of 5ml e-juice is an amazing number. Few small disposable pods have such a large pod capacity. HQD Cuvie Plus can last up to 1200 puffs. The concentration of nicotine is about 50mg, which is enough to eliminate your craving for nicotine. Provide a variety of flavor options, is perfect for vaping on the go.
HQD Cuvie Plus Disposable Vape,Disposable Vape HQD Cuvie Plus, 1200 Puffs Vape Pen
Nanning Goodman Technology Co.,Ltd , https://www.goodmentech.com