Principle
Isoelectric focusing gel electrophoresis is a technique for separating proteins based on the electrostatic charge or isoelectric point of a protein molecule. In isoelectric focusing, the protein molecules are 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 in which 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 can therefore 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 performed 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 fixation and dyeing
Reagents: acrylamide, bis-acrylamide, carrier ampholyte, 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. Glue
The formulation of the denatured isoelectric focusing gel of 8 cm x 7 cm x 0.75 mm pH 4-6 was prepared. Other pH ranges for 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 the urea, water, solution 1) and the carrier ampholyte solution to avoid vigorous shaking, and slightly heating the urea to dissolve 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 injected into the upper tank with the anode electrode solution before loading. Determine if there is any leakage.
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 large 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.
Isoelectric focusing gel electrophoresis is a technique for separating proteins based on the electrostatic charge or isoelectric point of a protein molecule. In isoelectric focusing, the protein molecules are 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 in which 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 can therefore 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 performed 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 fixation and dyeing
Reagents: acrylamide, bis-acrylamide, carrier ampholyte, 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. Glue
The formulation of the denatured isoelectric focusing gel of 8 cm x 7 cm x 0.75 mm pH 4-6 was prepared. Other pH ranges for 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 the urea, water, solution 1) and the carrier ampholyte solution to avoid vigorous shaking, and slightly heating the urea to dissolve 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 injected into the upper tank with the anode electrode solution before loading. Determine if there is any leakage.
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 large 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.
Garlic Flakes,Falksalt Wild Garlic,Crispy Garlic Flakes,Garlic Flakes For Sale
shandong changrong international trade co.,ltd. , https://www.cragriculture.com