Protein Technology Topics: Protein Isoelectric Focus Gel Electrophoresis

Principle

Isoelectric focusing gel electrophoresis is a technique that separates the electrostatic charge or isoelectric point of a protein molecule. In isoelectric focusing, the protein molecule is electrophoresed in a continuous and stable linear pH gradient formed by a carrier ampholyte. The carrier ampholyte is an aliphatic polyaminopolycarboxylic acid which forms a continuous pH gradient in the electric field where the positive electrode is acidic and the negative electrode is basic. A protein molecule carries a charge at a pH that deviates from its isoelectric point and therefore can move in an electric field; when the protein migrates to its isoelectric point, its static charge number is zero and no longer moves in the electric field, according to which Protein separation.

In isoelectric focusing, a good protein band resolution can be obtained only when high voltage is applied to both ends of the gel, which requires a very effective gel cooling system (otherwise it will lead to burning), ie gel simultaneous The heat transfer between the surrounding liquids is high. Due to the high heat transfer capacity of the flat gel and the convenient comparison of various protein samples at the same time, the flat glue is mostly used for isoelectric focusing.

Since isoelectric focusing is very sensitive to the difference in charge of proteins, for good repeatability, care must be taken when preparing protein samples to avoid any modification of the chemical composition and structure of the protein. In addition, protein-lipid, protein-protein interactions can cause charge changes, which in turn lead to isoelectric point migration or texture phenomena. Isoelectric focusing is usually carried out in a denaturing gel system containing urea unless it is specifically required to study protein-protein interactions or to maintain the biological function of the protein. Resolution can also be improved with non-ionic detergents.

2. Main instruments, reagents

Instruments: Microelectrophoresis systems, power supplies, syringes, containers for fixing and dyeing reagents: acrylamide, bis-acrylamide, carrier ampholytes, urea, ammonium persulfate, TEMED, Triton X-100, 2-mercaptoethanol, bromophenol Blue, phosphoric acid, sodium hydroxide, potassium chloride, trichloroacetic acid, Coomassie brilliant blue, methanol, acetic acid.
Stock solution: 1) 30% (w/v) acrylamide, 1% (w/v) bis-acrylamide; 2) 20% Triton X100; 3) 10% trichloroacetic acid; 4) 1% trichloro Acetic acid; 5) 1% bromophenol blue; 6) Coomassie brilliant blue staining solution; 7) Coomassie brilliant blue decolorizing solution;

3. Selection of carrier ampholytes

The carrier ampholyte is a mixture of polyaminopolyhydroxy compounds having a molecular weight of 600-900 Da having similar isoelectric points. The following is a formulation of a carrier ampholyte with different pH ranges of isoelectric focusing gel:

4. Potting

The formulation of a denatured isoelectric focusing gel of 8 cm x 7 cm x 0.75 mm pH 4-6 was prepared. Other pH ranges of isoelectric focusing can be configured with reference to the table.

Water: 5.4 ml; stock solution 1): 2.0 ml; carrier ampholyte solution pH 3.5-10 48 μl; carrier ampholyte solution pH 4-6 240 μl; ultrapure urea 6.0 g; 10% ammonium persulfate 25 μl; TEMED: 20 μl

Gluing step: 1) assembling the glue applicator; 2) mixing urea, water, solution 1) and carrier ampholyte solution to avoid vigorous shaking, slightly heating urea dissolves faster (with gloves, acrylamide is toxic); Add ammonium persulfate and TEMED, mix gently (start polymerization, operate quickly); 4) Pour the above mixed solution into a glue applicator (to avoid bubble generation); 5) Insert the comb and invade the comb into the glue (Do not generate bubbles); 6) Polymerize for about 1 hour; 7) Complete the polymerization, carefully remove the comb; 8) Connect the gel to the cooling device and insert it into the electric swimming pool. The protein sample is preferably injected with the anode electrode before loading. Determine if there is any leakage in the tank.

Note: 1) Wash the unpolymerized acrylamide at the bottom of the sample before loading, otherwise polymerization will occur during electrophoresis; 2) There are commercial isoelectric focusing gels, but only for horizontal plate electrophoresis systems (eg Pharmmacia-LKB, Hoefer).

Sample preparation and loading:
Generally, different thicknesses of gels, different number of comb holes will have different loadings, for example: 0.75mm thick, 15 holes of gel per hole maximum sample loading of about 15μl.

Denatured gel loading buffer 2 × Buffer (for pH 4-6): 1) urea: 2.4 g; 2) pH 3.5-10 carrier ampholyte: 20 μl; 3) pH 4-6 carrier ampholyte: 100 μl; 20% Triton X-100: 500 μl: 5) 2-mercaptoethanol: 50 μl; double distilled water: 1.7 ml; 1% bromophenol blue: 200 μl

5. Electrophoresis

Steps:

1) Mix the protein sample in an equal volume of 2× loading buffer, centrifuge at 10000×g for 5 min to remove the protein precipitate; 2) Add the protein sample to the bottom of the sample with a micro syringe, taking care not to spill. Note: For Coomassie Brilliant Blue staining solution, 10-30ug of protein-mixed crude extract (usually known as crude antigen) or 5-10ug of single protein fraction per lane is a reasonable loading concentration.

Electrophoresis fluid:

1) Add a cathodic electrophoresis solution to the upper tank (20 mM sodium hydroxide: freshly prepared from 1 M stock solution);

2) Add the anode electrophoresis solution (10 mM phosphoric acid: freshly prepared from 1 M stock solution) to the lower tank.

Isoelectric focusing electrophoresis conditions:

1) Connect the electrodes;

2) constant pressure 150V electrophoresis for 30 min;

3) Constant voltage 200V electrophoresis for 2.5h, the control current is about 10mA at the beginning, the current decreases during the focusing process, and the electrophoresis internal chamber temperature will rise to 40-50 degrees Celsius during electrophoresis.

6. Electrophoresis focusing post processing

Determine the pH gradient:

1) cutting the gel strip into small pieces of 0.5 cm or 1 cm;

2) soak each small piece of gel in 1 ml of 10 mM KCl for 30 min;

3) Measure the pH of the KCl solution,

Gel fixation:

1) Soak the gel in 10% trichloroacetic acid for 10 min;

2) Change to 1% trichloroacetic acid solution and continue to soak for at least 2h to remove the carrier ampholyte. Soaking overnight can better reduce the dyeing of the carrier ampholyte by Coomassie Brilliant Blue.

Gel staining: stained with Coomassie brilliant blue, then destained to make a dry gel.

7. Correction scheme for natural isoelectric focusing electrophoresis

If you want to perform natural isoelectric focusing electrophoresis, make some modifications;

1) No urea is added to the gel in the gel process;

2) Loading buffer (2×) 5ml: 1.8ml water, 200μl carrier ampholyte (same gelatin component), 3ml glycerin, mix in equal volume sample when loading, centrifuge 5min at 10000×g kind;

3) Electrophoresis at room temperature, connect the electrode, first 200V electrophoresis for 1.5h under constant pressure, and then electrophoresis for 1.5h at 400V.

8. Frequently asked questions and explanations

1) If a blurred band is produced, the focus is incomplete, which may be due to problems in electrophoresis or macromolecular proteins that limit their ability to migrate in the gel. If the focus time is too long or too short, the resolution of the strip will decrease. Increasing the voltage gradient can make the strip shape sharper. High molecular weight proteins can be better focused in agarose gels.

2) The skewed strip is usually checked for cleanliness due to an incorrect pH gradient, whether it is well linked to the gel, and the edge effect of the gel should be noted.

3) The texture phenomenon of protein bands is a problem often encountered in isoelectric focusing, which may be the following reasons:

a, protein aggregation or precipitation (especially near the isoelectric point), or the amount of loading is too large, 8M urea is usually used to prevent protein aggregation, detergents Triton X-100 and NP-40 are commonly used to prevent membrane protein aggregation, Therefore, it must be centrifuged to remove insoluble particles before loading.

b, residual nucleic acid in the sample, a variety of methods can be used to remove nucleic acid contamination, such as acid extraction, salt precipitation, nuclease digestion, etc.;

c, protein modification, isocyanate contaminants in non-ultra-grade urea may lead to carbamylation of proteins, pre-electrophoresis can remove isocyanates, protein sample processing or improper storage will occur including Cys residue oxidation a modification process such as deamination of an Asn or Gln residue;

d, the wavy strip is often due to the salt concentration in the sample is too high, sometimes the carrier ampholyte or electrode liquid is impure or the electrode is not clean will also produce wavy strips;

e, the electrode is not parallel to the gel connection. When the gel is configured, the reagent is impure. If the concentration of the carrier ampholyte is too low, the pH gradient may be uneven. If the pH gradient of the alkaline part of the gel is lost, the anode may drift. A carrier ampholyte supplemented with pH 9-11 or subjected to non-equilibrium pH gradient electrophoresis;

f, the high background of dyeing may be due to the carrier ampholyte remaining in the gel after fixation, increasing the fixation time of 1% trichloroacetic acid can be solved;

g, the loss or too light of the protein band may be due to the lower molecular weight of the protein (<10kDa> or the protein is not denatured during fixation, increase the concentration of trichloroacetic acid or use glutaraldehyde to fix;

h, complex protein mixtures can produce overlapping spots after isoelectric focusing, and changing the pH range of the isoelectric focusing gel can solve this problem. Further protein purification or immunoprecipitation treatment can avoid the generation of overlapping spots.

9. Other matters to be aware of

1) After isoelectric focusing, a dyed thin line (0.1mm) can be used to mark the position of the dye front;

2) There are subtle differences in the properties of the different electrolytes of the different brands. When using the carrier ampholyte gel from different sources, the protein separation pattern is slightly different. If you want to obtain the best repeatability, do not change the brand of carrier ampholyte;

3) Generally, a narrow range of pH gradients can improve resolution, but it takes more time, and a pH gradient of 2 is a better choice;

4) Due to the limitations of the power supply and gel cooling system, the longest gel length is 8-10 cm. In fact, electrophoresis of several different pH gradient gels is better than electrophoresis only for a longer gel.

5) When the isoelectric focusing time is long (>3000V·h), the cathode drift caused by the instability of the carrier ampholyte will become a serious problem, and the cathode drift will destroy the pH gradient, especially the gradient above pH8, in order to overcome this The problem was to develop a technique that performs isoelectric focusing electrophoresis in a short time (1600 V·h), ie, non-equilibrium pH gradient electrophoresis, which can focus proteins in a high pH range, but this method cannot be used to determine protein isoelectric points. .

6) Urea can promote protein solubilization and eliminate protein-protein and protein-lipid interactions, which can increase the focusing speed and resolution, while suppressing cathodic drift, but also note that the isoelectric point of denatured protein may be the same as natural protein. The difference.

7) If the protein solution contains SDS, urea can be added to a final concentration of 8 M. Under high concentration of urea, the interaction of SDS with the protein is minimal.

8) Add 2% Triton X-100 to the denaturing solution to ensure sufficient dissolution of the protein (especially membrane protein). Some authors recommend the use of zwitterionic detergents such as CHAPS and Zwittergent 3-14;

9) Ultra-thin gel (50-500um) is superior to common standard isoelectric focusing because of the high electric field strength and better cooling effect, resulting in faster focusing and higher resolution.

10) In polyacrylamide isoelectric focusing gels, high molecular weight proteins (>750kd) often exhibit abnormal migration behavior, and agarose gel or agarose-acrylamide gel is more suitable for high molecular weight proteins.

11) In Coomassie brilliant blue staining, the dyeing effect can be further improved by washing the gel with 0.25% SDS ethanol: acetic acid: water (33:10:57) solution after fixation with trichloroacetic acid for 10-30 min. The SDS can better remove the carrier ampholyte when combined with the carrier ampholyte.

10. Solid phase pH gradient isoelectric focusing electrophoresis

Introduction: Solid-phase pH gradient isoelectric focusing is an isoelectric focusing technique established in the 1980s. The medium used is some acrylamide derivatives with weak or weakly basic acids, which are in acrylamide and methylidene. Acrylamide has similar polymerization behavior. The double bond at one end of the immobilized electrolyte can be covalently bonded to the polyacrylamide medium in the polymerization, and the R group at the other end is a weak acid or a weak base, which can form a buffer system of weak acid or weak base in the polymer, and titrate with a buffer system. A pH range near the end point can be approximated as a linear pH gradient, so the difference between the solid phase pH gradient and the carrier ampholyte pH gradient is that the former molecule is not amphiphilic, and a pH gradient is formed during gel polymerization, without environmental field conditions. Changed and changed, the latter is an amphiphilic molecule that forms a pH gradient after migrating to its own isoelectric point in the electric field. Solid-phase pH gradient isoelectric focusing has higher resolution than traditional isoelectric focusing, and the larger the sample loading, the resolution can reach 0.001pH, which is one of the highest resolution electrophoresis methods.

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