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 often has a high concentration of salt in the sample, and sometimes the carrier ampholyte or the electrode liquid is impure or the electrode is unclean, and a wavy strip is also generated;
e, the electrode is not parallel to the gel connection. When the gel is configured, the reagent is impure. The concentration of the carrier ampholyte is too low, which may result in an unequal pH gradient. If the pH gradient of the alkaline part of the gel is lost, the anode may drift. Adding a carrier ampholyte of pH 9-11 or performing an unbalanced pH gradient electrophoresis;
f, the high background of dyeing may be due to the carrier ampholyte remaining in the gel after fixation, the fixed 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 precipitation 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 good 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 the gel cooling system, the long 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 8M. Under high concentration of urea, the interaction of SDS with 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 more advantageous than common standard isoelectric focusing because of the high electric field strength and better cooling effect, which makes the focusing speed faster and the resolution higher.
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 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, and a weak acid or weak base buffer system can be formed in the polymer, and the buffer system is used for titration. 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 an amphiphilic molecule, and a pH gradient is formed during gel polymerization, without environmental electric 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 current high resolution electrophoresis methods.
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 often has a high concentration of salt in the sample, and sometimes the carrier ampholyte or the electrode liquid is impure or the electrode is unclean, and a wavy strip is also generated;
e, the electrode is not parallel to the gel connection. When the gel is configured, the reagent is impure. The concentration of the carrier ampholyte is too low, which may result in an unequal pH gradient. If the pH gradient of the alkaline part of the gel is lost, the anode may drift. Adding a carrier ampholyte of pH 9-11 or performing an unbalanced pH gradient electrophoresis;
f, the high background of dyeing may be due to the carrier ampholyte remaining in the gel after fixation, the fixed 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 precipitation 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 good 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 the gel cooling system, the long 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 8M. Under high concentration of urea, the interaction of SDS with 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 more advantageous than common standard isoelectric focusing because of the high electric field strength and better cooling effect, which makes the focusing speed faster and the resolution higher.
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 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, and a weak acid or weak base buffer system can be formed in the polymer, and the buffer system is used for titration. 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 an amphiphilic molecule, and a pH gradient is formed during gel polymerization, without environmental electric 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 current high resolution electrophoresis methods.
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