Isolation, Purification and Identification of α
1
-AT
1
1
α
1
-antitrypsin, a glycoprotein of 51 kDa, is present in approximately equal concentrations in plasma and interstitial fluid. Its structure consists of 3 parts: 3
β-sheets (A-C), 9 α-helices (A-I) and an exposed mobile loop serving as the reactive center. Although there is some local tissue synthesis (e.g. in monocytes and macrophages), nearly all plasma α
1
-antitrypsin is synthesized by the hepatic parenchymal cells. Also referred to as serine protease inhibitor (α
1
-proteinase inhibitor or α
1
-Pi), it inhibits the serine proteases trypsin, chymotrypsin as well as pancreatic and especially granulocytic elastase. It forms tetrahedral complexes with the active sites of serine proteases, thus blocking their enzymatic active center.
Physiologically, this biological function is accomplished by the α
1
-AT released in the process of phagocytosis by polymorphonuclear leukocytes. The increased levels of α
1
- AT are common as it is an acute phase reactant. Its plasma concentrations rise several folds in the case of acute or chronic inflammation. The elevated levels are also seen in the late pregnancy and during oestrogen therapy because the synthesis of α
1
-antitrypsin is stimulated by oestrogens. The low levels of
α
1
-antitrypsin, on the other hand, are found in neonatal respiratory distress syndrome, severe neonatal hepatitis, preterminal disease of the pancreas and in severe protein losing enteropathies. A hereditary deficiency of α
1
-antitrypsin is associated with pulmonary emphysema and liver diseases including neonatal cholestasis (hepatitis), cirrhosis and hepatocellular carcinoma. However, the measured level of α
1
- AT does not drop below the normal range for these α
1
- AT- deficiency cases mentioned above. It is a slightly confusing. The truth is α
1
- AT can be released in the acute phase of inflammation to compensate the lose of the α
1
-
AT . Therefore, the level of α
1
- AT remains in the normal range for the α
1
-
AT-deficiency patient.
There are two main mutant variants of α
1
-antitrypsin, S and Z. The S mutation alone is not a serious threat to human health. However if it is inherited with the Z mutation, the compound heterozygote can lead to severely decreased plasma concentrations of α
1
-antitrypsin. ZZ homozygotes display an even more dramatic decrease in plasma concentrations of α
1
-antitrypsin, about an 85% deficit in relation to levels in WT individuals, and the deficiency of α
1
-antitrypsin in both
SZ and ZZ cases leads to proteolytic tissue damage and diseases such emphysema in the lungs. Other mutant forms of α
1
-antitrypsin are S iiyama and M malton.
The deficiency of α
1
-antitrypsin is due, not to the lack of its production, but rather to its aggregation in the endoplasmic reticulum. Specifically, in the Z variant, the
16

Isolation, Purification and Identification of α
1
-AT mutation is at the junction of the reactive center and the 5th strand of β-sheet A .
This mutation disrupts the salt bridge between Glu-342 and Lys-290 and causes the sheet to open. This conformation diminishes its activity. Studies are underway to counteract the effects of these mutations. Studies are underway to counteract the effects of these mutations.
Proteins are the most versatile macromolecules in living organisms and serve crucial functions in all biological processes. Protein functions are universal.
Therefore, it is extremely important to isolate, purify and identify the protein of your interest before research. Here we use α1-AT as an example to learn all the methods involved in the protein isolation, purification and identification for the following reasons. First, α1-AT has been well characterized. Secondly, all the procedures are suitable for current teaching practice, and can be applied to your future research equally.
This project consists of six experiments in three weeks. In the first week, the salting out method will be performed to make a primary isolation of proteins from the crude samples, and gel filtration chromatography will be used to remove the salts from the protein samples isolated in the previous salting out experiment. In the following week, DEAE-cellulose ion exchange chromatography is applied for further purification, and then the purified protein will be quantified and its enzymatic activity will be determined. In the third week, the SDS-polyacrylamide gel electrophoresis (SDS-PAGE) will be used to identification on the molecular size.
17

Isolation, Purification and Identification of α
1
-AT
【 Objective 】
1.
To learn and understand the concepts and principles of protein precipitation
(salting-out) method for protein isolation.
2.
To become familiar with the procedure of the protein precipitation method.
3.
To become able to isolate α
1
-antitrypsin primarily from human serum using the protein precipitation method.
【 Principle 】
The solubility of proteins depends on, among other things, the salt concentration in solution. At low concentration, salt stabilizes the charged groups on a protein molecule, thus enhancing the solubility of proteins in solution. This is commonly known as salting-in . As the salt concentration increases, the protein solubility increases accordingly (in most cases) and then reaches the maximum point. Higher salt concentration means that less water is available to solubilize proteins. Therefore, as the salt concentration continues to increase, protein molecules start to precipitate since there are no enough water molecules to interact with proteins. This phenomenon of protein precipitation in the presence of excess salt is known as salting-out.
In this experiment, either a saturated salt solution or powdered salt crystals are slowly added to the protein solution to bring the salt concentration up gradually.
For example, the salt concentration is 25% saturated when 3.0 ml of the salt-free protein solution is mixed with 1.0 ml of the saturated salt solution; 50% when mixed with 3.0 ml of saturated solution and 75% when mixed with 9 ml of saturated solution.
Since different proteins may differ in their charged groups, they could be precipitated at different salt concentrations. The partial collection of the precipitated proteins at different salt concentrations is referred to as fractionation .
【
Experimental Materials
】
1.
Equipment and tools


Graduate cylinder
Balance


Centrifuge
 Centrifuge tube
 Filtration devices (filter pump, filter flask ， funnel, rubber stopper )
Beakers
18

Isolation, Purification and Identification of α
1
-AT
 Glass rod
 Refrigerator
2.
Reagents
 Human serum, 15ml


Saturated (NH
4
)
2
SO
4
solution, 15ml
 (NH
4
)
2
SO
4
crystals
0.05M, pH6.4 Phosphate buffer solution (PBS)
【 Procedure 】
1.
First fractionation
Measure 15 ml of human serum and 15 ml of saturated (NH
4
)
2
SO
4
solution with graduate cylinders
， respectively. Pour the latter into the former. Mix them together in a beaker thoroughly with a glass rod. At this moment, the
(NH
4
)
2
SO
4
concentration reaches 50% saturation. Proteins of large molecular weight in human serum may be precipitated at this salt concentration, such as
α2-globulin, β-globulin, γ-globulin and so on. α1-AT is a kind of α1-globulin.
It isn’t precipitated. Store the solution at 4℃ for 20 minutes.
2.
Centrifugation
Transfer the solution to centrifuge tubes. Balance the centrifuge tubes by pair.
Centrifuge at 3000 rounds per minute for 20 minutes.
3.
Second fractionation
Take out the centrifuge tubes from the centrifugal rotor gently when it stops completely. Two layers in the tube can be seen. 【 Note: Never shake the tubes and disturb the flurry bottom layer.
】 The top layer is a transparent solution, and the bottom is the protein precipitates. 【 Note: α1-AT is in the solution.
】 Pour the top layer into a cylinder carefully and measure the volume. Add an adequate amount of (NH
4
)
2
SO
4
crystals into the solution in the ratio of 17.6 g of (NH
4
)
2
SO
4
crystals per 100 ml solution. Then stir up to dissolve (NH
4
)
2
SO
4
crystals completely. Store the solution at 4℃ for 20 minutes. 【 Note: To accelerate the crystal dissolution, you can use the glass rod to crash the crystals into small pieces. The (NH
4
)
2
SO
4
concentration now can be as high as 75%. α
1
-AT, along with some other kinds of proteins, in human serum will be precipitated.
】
4.
Filtration
Cut two pieces of round filter paper to match the inner diameter of the funnel.
The size of the filter paper should be equal to or smaller than the inner diameter of the funnel. Place these two pieces of filter paper in the funnel. Place the rubber stopper of the funnel on the mouth of the filter flask tightly. Connect the filter flask to the filter pump with a rubber tube. Rinse the filter papers
19

Isolation, Purification and Identification of α
1
-AT with solution, which makes the filter paper wet to stick on the funnel. Pour the solution into the funnel slowly and then turn on the filter pump. The liquid in the solution will be sucked into the flask and leave the precipitate on the filter paper. Continue to suck the liquid out until the precipitate on the filter paper becomes paste-like material. Turn off the filter pump.
5.
Transfer the precipitate carefully to a beaker with a spatula.
6.
Add 2 ml 0.05 mol pH6.4 PBS in the beaker to dissolve the precipitate. This solution contains large quantity of α
1
-AT isolated from human serum.
【 Note: When performing this experiment, the glass rod, beakers and all the glassware should be kept dry to ensure the complement of this experiment.
】
20

Isolation, Purification and Identification of α
1
-AT
【 Objective 】
1.
To learn and understand the principles of desalting method for protein purification.
2.
To become familiar with the experimental procedures of desalting method.
3.
To become able to remove the salts in protein solution.
【 Principle 】
The technique of gel filtration (exclusion chromatography, gel chromatography, molecular sieve chromatography) was developed in 1960’s.
Gel filtration chromatography separates different proteins, peptides, and even oligonucleotides on the basis of the sizes of the molecules to be separated. Gel is composed of porous beads. When molecules move through a column of porous beads, they can diffuse into the beads to a certain degrees. Smaller molecules diffuse into the pores of the beads deeper and, therefore, move through the column more slowly, whereas larger molecules enter less or not at all and then flow through the column more quickly. This is the molecular sieve effect. As a result, larger and smaller molecules move through the column in different retention time.
Both molecular weight and three dimensional shape affect the degree of retention.
Gel filtration chromatography, thus, can be used for analyzing molecular size, for separating components in a mixture, or for removing salts from macrobiomolecule precipitates.
Take protein purification as an example. When a mixture of proteins is added to a column, they will flow through the column driven by a constant flow of a buffer solution. As the proteins flow through the column, the larger ones will be excluded from the beads, flow swiftly around the beads and thus quickly exit the the column. Meanwhile, the smaller ones are easy to enter the pores, and after passing through many pores on many beads, they will get out there eventually. A better analogy is a system of lava rocks, water, and marbles. Imagine packing a wide tube with lava rocks, and what would happen if you add to the top end of the tube a bucket of water mixed with marbles? Since lava rock is porous, you would expect the water to enter into it and trickle slowly down to the bottom of the tube.
The marbles, on the other hand, would be too big to enter the pores. Instead, they would roll quickly along the outsides of the rocks, exiting the column first. In this experiment, the gel beads are the lava rocks, the small proteins the water, and the large proteins the marbles.
21

Isolation, Purification and Identification of α
1
-AT
【 Procedure 】
1.
Set up the column
Clamp the column tube vertically. Keep the outlet of the column tube closed.
Add PBS (0.05M, pH 6.4) to the 1/3 volume of the column tube. Place a plastic beaker below the outlet of the column.
2.
Pack the column
Stir up the pre-prepared gel solution in a beaker carefully using a glass rod, mix the gel solution gently. 【 Note: Clean the rod before use or it will create carry-over contamination.
】 Add the gel solution into the column to about 2/3 volume of the column. When a clear layer about 1cm high appears on the top of the opaque gel solution (or 1 cm high of gel sediment forms at the bottom of the gel column), open the valve of the outlet and drain the solution slowly.
Control the flow speed at 30 drops/minute until the height of the gel column reaches the 1/3 – 1/2 of the column height. 【 Note: (1). When packing the column, keep the stable flow speed, to ensure to get a stable and uniform column. (2). Leave the PBS solution 2-3 cm above the gel column to avoid being dried due to evaporation. (3). The height of the gel is the height of the column after the gel fall down completely.
】
3.
Balance the column
Connect the outlet of a flask to the column in a rubber tube. Pour PBS (0.05M, pH 6.4) into the flask. Adjust the clamp on the rubber tube to balance the flow-in speed of PBS into the column and flow-out speed at the outlet of the column. Measure the pH of the flow-out solution. Close the outlet when the pH at the inlet and the outlet of the column are equal. 【 Note: the gel material is very expensive, so we reuse it. The gel in this experiment might be used previously, and could be contaminated by ammonia sulfate. The contaminated gel can influence the purification quality. Therefore, you need to use Nessler reagent to determine the presence of ammonium ions before adding the sample.
】
4.
Add samples
Suck out excess volume of the PBS solution on the top of the gel column using a Pasteur pipette (or dropper), and immediately add the sample solution onto the gel column slowly by draining the sample solution along the inner wall of the column tube. Open the outlet and control the flow speed at 30 drops/minute.
When the sample solution enters the gel column completely, wash the inner wall of the column tube with a small amount of PBS. 【 Note: Adding sample solution is time-consuming. Be patient. Do not add the sample solution directly onto the top of the gel column to avoid disturbing the flat surface of the column.
】
22

Isolation, Purification and Identification of α
1
-AT
5.
Collect the eluted sample
Add 2-3 ml PBS onto the top of the gel column. Connect the flask to the gel column using a rubber tube and begin to wash the gel column. As PBS flows, you can observe a yellowish band moving downward on the gel column, which is the protein sample. When the yellow band reaches the bottom, take a small amount of the eluate to react with Biuret reagent. If the color of the reagent turns to purple, it indicates the protein is present in this eluate. Begin to collect the eluted solution. Continue to collect the eluate until the Biuret reagent does not show the purple color.
6.
Wash the gel column
Wash the column with PBS at full speed. Test the collected portion with Nesslar reagent. This reagent will turn to orange color once reaction with ammonia sulfate present in the gel beads. Wash the gel column until the reaction color disappears.
7.
Reclaim the gel materials
Turn the column tube upside down. Put the mouth of a pipette bulb to the small exit end of the column and squeeze the bulb to flush the beads out to the beaker.
8.
Measure and record the total volume of the eluted sample. Take 1.0 ml into a small tube for future analysis. Transfer the remaining into a big plastic test tube, and keep them in refrigerator.
23

Isolation, Purification and Identification of α
1
-AT
【 Objective 】
1.
To learn and understand the principles of ion exchange chromatography.
2.
To become familiar with the experimental procedures of protein purification using ion exchange chromatography.
3.
To become able to remove non

1-AT components in the crude protein sample.
【 Principle 】
Ion exchange chromatography separates molecules based on the differences in the overall charge of proteins. It is commonly used for protein purification as well as for purification of oligonucleotides, peptides, or other charged molecules. The protein to be purified must have charges opposite to that of the functional groups on the resin. For example, immunoglobulins, which generally have an overall positive charge, will bind well to cation exchangers, which contain negatively charged functional groups. Ion exchange containing diethylaminoethyl (DEAE) or carboxymethyl (CM) groups are most commonly used for this purpose. For example, DEAE is of positive charges, so it can absorb proteins that are negatively charged.
CH
2
CH
2
NH
+
C
2
H
5
CH
2
C
O
O
-
C
2
H
5
DEAE-cellulose ion exchanger CM-cellulose ion exchanger
Since this interaction is ionic, binding must take place under low ionic conditions. Desorption is then brought out by increasing the salt concentration or by altering the pH of the mobile phase.
The ionic properties of DEAE and CM are dependent on pH. Both work well as ion exchangers in the pH range of 4 to 8 where most protein separations take place. The property of a protein which governs its adsorption to an ion exchanger is the net surface charge. Since the surface charge is the net result of weak acidic and basic groups of a protein, separation is highly pH dependent. Going from low to high pH, the surface of a protein shifts from positively charged to negatively charged. The pH vs. net surface curve is an individual property of a protein, and
24

Isolation, Purification and Identification of α
1
-AT constitutes the basis for selectivity in ion exchange chromatography. At a pH value below its isoelectric point a protein (that is, positive charged) will adsorb to a cation exchanger such as one containing CM groups. Above the isoelectric point protein (that is, negatively charged) will adsorb to an anion exchanger such as one containing DEAE groups.
Desorption can be brought out by varying pH or ionic strength. When pH value changed, the adsorbed protein has passed its isoelectric point and the surface charges altered too. Thus, varying the pH of the mobile phase can separate and collect the target protein.
At low ionic strengths, all components having binding affinity for the ion exchanger will be tightly adsorbed at the top of the ion exchanger and nothing will be in the mobile phase. Then the ionic strength of the mobile phase will be gradually increased by adding a neutral salt, the salt ions will compete with the protein, and more adsorbed proteins will be partially desorbed and start moving down the column. The more net charges a protein has, the higher the ionic strength is needed to bring about the desorption. In order to optimize protein separation in ion exchange chromatography, a pH value is carefully chosen that creates sufficiently large net charge differences among the different proteins components.
【 Procedure 】
1.
Set up and pack the column
Follow the procedure described in the section of Gel Filtration Chromatography
(Eexperiment 2). The minor difference is that the height of the gel column for ion exchange chromatography is 1/3 of the column height 【 Note: Not 2/3 in
Experiment 2 】 . Equilibrium the column with 0.05 M pH 6.4 PBS. Connect the exit hole of a flask to the column in a rubber tube. Pour PBS (0.05 M, pH
6.4) into the flask. Adjust the clamp on the rubber tube to keep the flow speed of PBS into the column and out of column at the exit end. Measure the pH of the flow-out solution. Close the exit end when the pH at the entrance and the exit are equal. 【 Note: The equilibrium step is very important due to the principle of the DEAE-cellulose ion exchange chromatography. Because pH can influence adsorption and desorption of proteins and the
DEAE-cellulose gel is alkalic, the equilibrium time will be a slightly long.
】
2.
Add sample
Same as that in the section of Gel Filtration Chromatography (Eexperiment 2).
3.
Open the exit end of the column tube and control the flow speed at 1 drop/4 second.
【
Note: Control the speed to allow the protein adsorb to the
DEAE-cellulose enough.
】
4.
Set up a reaction plate for color comparison.
25

Isolation, Purification and Identification of α
1
-AT
5.
Periodically transfer one drop of the eluate to the hole of pre-prepared reaction plate. If the color of the reagent turns purple, it indicates that protein is present in this eluate. But it is the flow-through proteins not the

1-AT component of our interest since it does not bind to the ion exchanging column.
Elute continuously until no more protein can be detected. 【 Note: Do not discard this portion. Due to many personal reasons, the proteins of interest could be present in this portion. If you can’t collect the proteins of interest in the next step, this portion will be used in the later step.
】
6.
Use the elution buffer of 0.12 M pH 6.4 PBS to wash out the proteins adsorbed on the column at full speed. Use the Biuret reaction to determine the collecting point. When the color of the reagent turns purple, immediately collect the eluted solution.
【
Note: This collection should be as quick as possible since the proteins adsorbed on the lower portion of the column could be eluted out very fast. Collect all the eluate until the reaction does not show any color change. Measure the eluate volume with a cylinder.
Transfer it into the plastic centrifugal tubes. Mark the tubes and keep them in refrigerator.
】
26

Isolation, Purification and Identification of α
1
-AT

【 Objective 】
1.
To learn and understand the principles of protein activity determination.
2.
To become familiar with the procedures of measuring the protein activity.
3.
To become able to determine the α
1
-antitrypsin activity using spectroscopic measurement.
【 Principle 】
The activity of

1-AT is determined by measuring its capacity of inhibiting the enzymatic activity of trypsin.
Trypsin is an enzyme hydrolyzing peptides. One of its substrates is
N-benzoyl-L-arginine-p-nitroanilide (BAPNA), a simple peptide. Enzyme trypsin will cleave BAPNA to liberate p-nitroaniline.
H
2
N C
H
N
H
2
C
H
2
C
H
2
C c
NH
CH
NH C
OH
O
O NH
NO
2
N-benzoyl-L-arginine-p-nitroanilide (BAPNA)
Trypsin
BAPNA + H
2
O -------------------

N-Benzoyl-L-arginin + p-Nitroanilin p-Nitroaniline is of yellow color whose spectrum is different from BAPNA.
The formation of the hydrolytic product, therefore, can be conveniently identified using a simple spectroscopic measurement.

1-AT has been known as an inhibitor of trypsin to inhibit the trypsin activity partially or completely.
Therefore, the activity of

1-AT can be determined indirectly by comparing the trypsin activity in the presence or absence of

1-AT.
27

Isolation, Purification and Identification of α
1
-AT
【 Experimental Materials 】
1.
Test tubes
2.
BAPNA, Trypsin, 33% HAC, Tris-HCl buffer, serum, the samples collected through the experiment above
3.
722-spectrometer
4.
Incubator
5.
Cylinder, transporter etc.
【 Procedure 】
1.
Prepare 5 eppendorf tubes and mark them. Add the following reagents in each marked tube accordingly. The quantity of each reagent is given in the table below.
2.
Use 0.1 M Tris-HCl buffer, pH 8.0 containing 0.01M CaCl
2
to adjust the final volume of each reaction mixture to 1.0 ml.
Volume: ml
Tris buffer
Serum
G-25
DEAE
Trypsin
#1 blank #2 control #3 serum #4 G-25 #5 DEAE
1.0 0.5 0.4 0.4 0.4
- - 0.1 - -
-
-
-
-
-
0.5
-
-
0.5
0.1
-
0.5
-
0.1
0.5
3.
Incubate the test tube at 37℃ for 5 minutes.
4.
Add 2.5 ml of freshly prepared BAPNA to each tube.
5.
Incubate at 37℃ for 10 minutes.
6.
Stop the reaction by adding 0.5 ml of acetic acid (33% v/v) in each tube.
7.
Record the OD readings at 410 nm of each reaction mixture. Calculate the activity of each mixture. The full enzymatic activity is determined based on the readings of the #2 tube in which no inhibitor is added in the reaction mixture. The #1 tube is used for the blank reading on spectrophotometer.
28

Isolation, Purification and Identification of α
1
-AT
【 Objective 】
1.
To learn and understand the principles of Biuret method for protein quantitation.
2.
To become familiar with the procedures of protein quantification.
3.
To become able to use the Biuret method to quantify the purified

1-AT.
【 Principle 】
Under alkaline conditions, substances containing two or more peptide bonds can form a purple complex with copper salts in the reagent.
【 Experimental Materials 】
1.
Test tubes, cylinder and transporter etc.
2.
Biuret reagent, 0.9% NaCl, serum, samples collected through the experiment above
3.
722-spectrometer
【 Procedure 】
1.
Add the following reagents in each marked tube accordingly. volume: ml
0.9% NaCl
Serum
G-25 sample
DEAE sample
Biuret reagent
#1 blank
0.1
-
-
-
5.0
#2 serum
-
0.1
-
-
5.0
#3 G-25
-
-
0.1
-
5.0
#4 DEAE
-
-
-
0.1
5.0
2.
Mix well and incubate at room temperature for 30 minutes.
3.
Record the OD readings at 520 nm, calculate the concentration of the samples using the formulation below. (Please see the details about Biuret method in Experiment 1.)
Cu=Au/As×Cs
29

Isolation, Purification and Identification of α
1
-AT
【 Objective 】
1.
To learn and understand the principles of SDS-PAGE method.
2.
To become familiar with the procedures of protein separation.
3.
To become able to separate proteins using SDS-PAGE method.
【 Principle 】
Electrophoresis is the migration of charged molecules in solution in response to an electric field. The migration rate of the charged molecules depends on the strength of the electric field; the net charge, size and shape of the molecules as well as the ionic strength, viscosity and temperature of the medium in which the molecules are moving. As an analytical tool, electrophoresis is simple, rapid and highly sensitive. It is used as an analytical method to study the properties of a single charged species, and as a separation technique.
1.
Support matrices
Generally the sample is run in a support matrix such as paper, cellulose acetate, starch gel, agarose or polyacrylamide gel. The matrix inhibits convective mixing caused by heating and provides a record of the electrophoretic run: at the end of the run, the matrix can be stained and used for scanning, autoradiography or storage.
In addition, the most commonly used support matrices - agarose and polyacrylamide - provide a means of separating molecules by size, in that they are porous gels. A porous gel may act as a sieve by retarding, or in some cases completely obstructing, the movement of large macromolecules while allowing smaller molecules to migrate freely. Because dilute agarose gels are generally more rigid and easy to handle than polyacrylamide of the same concentration, agarose is used to separate larger macromolecules such as nucleic acids, large proteins and protein complexes. Polyacrylamide, which is easy to handle and to make at higher concentrations, is used to separate most proteins and small oligonucleotides that require a small gel pore size for retardation.
2.
Continuous and discontinuous electrophoresis
There are two types of buffer systems in electrophoresis, continuous and discontinuous. A continuous system has only a single separating gel and uses the same buffer in the tanks and the gel. In a discontinuous system, a non-restrictive large pore gel, called a stacking gel, is layered on top of a separating gel called a resolving gel. Each gel is made with a different buffer, and the tank buffers are
30

Isolation, Purification and Identification of α
1
-AT different from the gel buffers. The resolution obtained in a discontinuous system is much greater than that obtained with a continuous system. The discontinuous system means: (1) The gel is discontinuous; (2) The buffer (pH and ionic strength) is discontinuous; (3) The electric gradient is discontinuous.
3.
Separation of proteins
Proteins are amphoteric compounds; their net charge therefore is determined by the pH of the medium in which they are suspended. In a solution with a pH above its isoelectric point, a protein has a net negative charge and migrates towards the anode in an electrical field. Below its isoelectric point, the protein is positively charged and migrates towards the cathode. The net charge carried by a protein is in addition independent of its size - ie: the charge carried per unit mass (or length, given proteins and nucleic acids are linear macromolecules) of molecule differs from protein to protein. At a given pH therefore, and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both size and charge of the molecules.
4.
Separation of proteins under denaturing conditions
Sodium dodecyl sulphate (SDS) is an anionic detergent which denatures proteins by "wrapping around" the polypeptide backbone - and SDS binds to proteins fairly specifically in a mass ratio of 1.4:1. In so doing, SDS confers a negative charge to the polypeptide in proportion to its length, i.e.
the denatured polypeptides become "rods" of negative charge cloud with equal charge or charge densities per unit length. It is usually necessary to reduce disulphide bridges in proteins before they adopt the random-coil configuration necessary for separation by size: this is done with 2-mercaptoethanol or dithiothreitol. In denaturing
SDS-PAGE separations therefore, migration is determined not by intrinsic electrical charge of the polypeptide, but by molecular weight .
5. V=EQ/6
r
There are factors of three aspects to decide the velocity of the charged molecules.
: E
: Q, r ,(shape)
: η
31

Isolation, Purification and Identification of α
1
-AT
【 Procedure 】
1.
Preparation of the gels: Resolving (Separating) Gels and Stacking Gels
volume: ml
Reagent
30% Acr-Bis
1.5M Tris-HCl pH 8.8
0.5M Tris-HCl pH 6.8
H
2
O
Glycerol
10% SDS
Peroxydisulphate
100mg/ml
TEMED
10% separating gel(ml)
10
7.5
-
6.1
6.0
0.6
0.15
4% stacking gel(ml)
1.4
-
2.4
6.3
-
0.2
0.2
0.04 0.02
【 Note: (1) Peroxydisulphate needs to be prepared freshly. (2)
Peroxydisulphate and TEMED will be added just before pouring the gel mixture. (3) The polymerization is initiated by adding fresh 10% ammonium persulfate to the mix followed by N,N,N',N'- tetramethylethylenediamine
(TEMED)-accelerator. The amounts of each catalyst depend on the quality of acrylamide used, and should be determined in advance by trial and error.
Once the catalysts are added, polymerization occur quickly. (4) Immediately after pouring the separating gel of the gel, add enough amount of water to cover the gel with 1 cm high of water solution. The step is to form a smooth and completely leveled surface on top of the separating gel, so that protein bands are straight and uniform. (5) After pouring the stacking gel mix, insert the comb. When the gel is formed, remove the comb, fill with electrophoresis buffer.
】
2.
Prepare samples
3.
Prepare the samples to a uniform concentration of 2mg/ml. Then take 1 ml of each sample and mix with 1ml of sample buffer each. Heat the mixture for 5 min at 100℃ in a "float" or in a water-bath.
4.
Load the samples
5.
Layer samples under buffer on stacking gels. 20μl each pore.
6.
Electrophoresis: 100V 2-3 hours
7.
Stain the gels: incubate the gel in staining buffer for 20 minutes.
8.
Decolor the gels: Immerse the gel in the decoloring solution and incubate overnight until the gel becomes clear and transparent.
9.
Dry the gels
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