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Storage:RT，4 years
A-50 Product Profile
DEAE-SephadexA-50(Diethylaminoethyl glucan A-50) is A weakly basic anion exchanger. Acid protein can be adsorbed by converting CL- type into OH- type with NaOH. The gamma globulins in serum belong to neutral proteins (isoelectric PH6.85-7.5), and the rest belong to acidic proteins. The acid proteins were adsorbed by DEAE-Sephadex A-50 in pH 7.2-7.4, only γglobulin was not adsorbed. Thus, by column chromatography gamma-globulin can be eluted first in elution, while other proteins are adsorbed on the column, and purified IgG can be isolated.
Product parameter
Product name: DEAE-Sephadex A-50
Substrate: Sephadex
Particle size: 40-120μm
Maximum flow rate: 45cm/h
Working pH: 2-9
Operating temperature: room temperature
Storage condition: room temperature
A-50 Experimental Procedure
1. Pretreatment
DEAE-Sephadex A-50(hereinafter referred to as A-50)5 grams, suspended in 500ml of distilled water, after 1 hour pour the top fine particles.
At the ratio of A-50 plus 0.5M NaOH 15ml per gram, A-50 was soaked in 0.5M NaOH, stirred well, left for 30 minutes, pumped into A Brinell funnel (padded with 2 layers of filter paper), and repeatedly pumped with distilled water until the PH was neutral. furthermore
0.5M HCl is processed in the same operation process as above, and finally 0.5M NaOH is processed again. After treatment, soak A-50 in 0.1M, PH 7.4PB overnight.
2. Install the column
(1) The chromatographic column is fixed vertically on the titration iron frame, the bottom of the column is padded with a garden of nylon yarn, and the water outlet is connected with a latex or plastic tube and closed
Switch.
(2) Pour 0.1M, PH7.4PB into the column along the glass rod to a height of 1/4, then pour into the pre-treated and adjust with the buffer solution as above
To A thin paste of A-50. When A-50 gel settles 2-3cm thick, open the spiral clamp of the water outlet, control the flow rate of 1ml/ min, and continuously pour the paste A-50 gel to the required height.
(3) Close the water outlet, after A-50 gel has completely settled, put A circular filter paper on the cylinder surface, and plug the upper mouth of the column with a rubber plug
The needle inserted into the rubber plug and the attached latex or plastic tube are connected to the eluent bottle.
3. Balance
Open the outlet screw clamp, control the flow rate of 12-14 drops/min, so that about 2 times the bed volume of eluent outflow. The PH value and ionic strength of eluent and effluent were determined by PH meter and conductance meter respectively. When consistent, close the water outlet and stop the balance.
4. Sample addition and elution
Open the upper rubber plug and the lower spiral clamp, so that the liquid in the column slowly drips out. When the cylindrical liquid is tangential to the cylindrical surface, the water outlet is immediately closed, and the sample is added along the wall of the column with a capillary dropper (the volume should be < 2% of the bed volume, and the protein concentration should be <100mg). Loosen the spiral clamp of the outlet to make the cylinder sample slowly enter the column, and immediately close the lower mouth when it is in contact with the cylinder surface, and wash the column wall with a small amount of eluent for 2-3 times; Then release the water outlet, so that the wash liquid into the bed column, and then immediately add several ml eluent on the cylinder surface, tightly plug the upper port of the column, so that the entire elution process into a closed system. And control the flow rate of 12 ~ 14 drops/min.
5. Collection
Collect in test tubes at the beginning of elution. Collect 3 ~ 5ml per tube. Collect 10 to 15 tubes.
6. Measure protein
The OD280nm and OD260nm of each tube were determined by 751-G type ultraviolet spectrophotometer, as described above
The content of tubulin was calculated by the formula. The elution curve was drawn with OD280nm as the vertical coordinates and test tube number as the horizontal coordinates.
7. Merge and concentrate
The tubes of the upper and lower sections of the eluting peak were combined and washed to the required volume with PEG(MW6000), and 0.02% NaN3 preservative was added
Store at 4℃for later use.
8. Regeneration of A-50 gel
The column was washed with 2M NaCl to OD280nm<0.02 of the effluent, and then washed with distilled water to remove the salt. Then the A-50 is processed again according to the pretreatment process to achieve regeneration. Recently used to soak in elution slow
Store at 4℃in the flush; If not used in the near future, wash with anhydrous alcohol twice, then dry in a 50℃temperature box, bottle and store.
(4) Precautions
1. Column selection: Theoretically, as long as the column is long enough, the ideal resolution must be obtained, but the flow rate of the chromatographic column is the same
As the pressure gradient is related, the flow rate slows down, the peak widens and the resolution decreases as the column length increases. As the diameter of the column increases, the non-uniformity of liquid flow increases and the resolution decreases obviously.
2. The purification process must strictly control the PH and ionic strength of the debuffering solution. Samples with A-50 gel must be cushioned with elution
Column chromatography can be performed only after the liquid is completely balanced.
3. The installed column bed must have a flat surface, no trench flow and bubbles, otherwise it should be reinstalled.
4. The flow rate should be strictly controlled during elution, and not too fast.
5. The sample size should be small, and the concentration should not be too high.
6. Prevent cylinder surface from drying out during sample addition and entire elution process.
Note：Product information may be optimized and upgraded. Please refer to the actual label information for accuracy.
