Separation purification and identification of human serum antitrinase (alpha1-AT).

-related topics Analysis of protease activity determination Focused protease research new progress "Objective Requirements"
1. Mastering the principles, operation and identification methods of the steps of alpha 1-AT separation purification 2. Mastering basic technical operations and instrument use 3. Familiar with the classification method of human
serum
protein
and understanding the properties of alpha-AT4. Separation purification type 5. Learn to use the learned separation purification method for basic scientific research design 6. Practice research paper basic writing"Teaching Content I"
segmented salt analysis method, gel
filtration
method (salt analysis method concept and principle, segmented salt analysis concept, molecular sieve effect, columnal analysis basic technology)Experimental principles
1. Salt analysis method is a high use of protein in the A method of separation with different degrees of solubility in a salt solution. By adding ammonium sulfate to the protein solution, the surface charge of the protein is normalized and the hydration membrane is destroyed, causing the stability factor of the protein in the aqueous solution to be removed and precipitated.
2. The method of changing the salt concentration by segmentation for separation purposes is called segmented salt analysis.
gel filtration, also known as molecular sieve layering. When the mixture flows with the flow phase through a layer column equipped with a gel as a fixed phase, the material with a large molecular weight is first washed away along the gap between the gel particles because it cannot enter the gel mesh hole (small resistance, short process, large flow rate), and the substance with a small molecular weight is blocked by being able to enter the gel mesh hole, the flow rate is slow, the process is long and then washed away, because the flow rate can be different sizes of molecules separated.Instruments and
Reagents
1.
1.
centrifuges
. 2.
vacuum pump
3. Saturated ammonium sulfate solution: 767g solid ammonium sulfate, added 1000ml of water,
heat
to dissolve it, cool at room temperature after 4 degrees C to stay overnight, and then use ammonia to adjust pH to neutral. 4. Solid (NH4)2SO45. Sephadex G-256. Nye Reagent 7. Double shrink reagent 8.pH7.4, 0.05M PBS(Step)A segment of salt analysis 1. Watch the separation and identification of human serum antitrypsin (alpha1-AT) teaching video 2. Take 20 ml of serum, pour it into a clean be cup, slowly add 20 ml of saturated ammonium sulfate, stir side by side, put in a 4c refrigerator for 30 minutes 3. Remove the beogroup from the refrigerator, pour the serum into the centrifuge tube, 3000rpm.
centrifugation 20 minutes 4. Pour the upper clean into a clean barrel, record the volume, pour into a clean beech, according to 17.6g/100ml to take solid ammonium sulfate, slowly add to the upper clear, while stirring. 30 minutes in the refrigerator 5. Pour the liquid in the beo cup into the filter, negative pressure pumping filter 6. Scrape off the coarse protein on the filter paper, dissolve with 4 ml buffer, ready to add samplesII gel filtration analysis gel column preparation: (1) connecting the layer column (2) Clamp the outlet, add 1/3 volume of 0.05M pH6.4 PBS, pour, wait for the gel to naturally subside about 1cm, open the outlet, control flow rate, 60 drops / minute (3) after filling the column, connect the lower port bottle, adjust the flow rate, so that the inlet and outlet flow rate is the same.
Balance to the inlet pH and the same value of the outlet pH (i.e. pH test paper color is the same), close the outlet, wait for the addition of 2. sample: use a straw to absorb the buffer above the glue surface, immediately add the protein coarse liquid (slowly join along the pipe wall) 3. Open the outlet, transfer speed 15 drops / minute, to the protein coarse liquid completely into the glue surface, with a small amount of buffer to clean the pipe wall.
the lower port bottle 4. Detection: detection of semen with double shrink reagent. Wait for the reagent to turn red and start collecting until the reagent does not change color to stop collecting. Measure and record the volume of the collection fluid, leave 1 ml of samples (labeled G-25 samples), put them in a freezer tube and freeze in the refrigerator ; the remaining liquid is collected in a large test tube, marked, and the next experiment is done with 5. Wash ammonium salt: full-speed wash-out, test the washide with Nye reagents, until the orange disappears, recycle the gel
"Note"
1. Salt The utensils used for analysis must
cand
2. When salting, saturated ammonium sulfate or solid ammonium sulfate should be added to the serum, and side-by-side stirring 3. Layering column should be left with a small amount of liquid, to avoid exposing the gel to the air 4. gel filtration sample, so that the sample along the pipe wall slowly flow, do not break the gel surface
.