SDS-PAGE problems and common problems analysis

What's the principle of lamination (or concentrate)? Weak acids in buffer system, such as glycine, seldom dissociate at ph6.7, and their effective swimming rate is very low, while chloride ions have a very high swimming rate, and the swimming rate of protein is between them When the voltage is applied, the chloride ion as the lead ion and the glycine ion as the tail ion are separated, and a zone with low conductivity is left behind Because the conductivity is inversely proportional to the electric field strength, a higher voltage gradient will be obtained in this zone, which will accelerate the swimming of glycine ion, make it catch up with chloride ion, and establish a stable state (I don't quite understand this sentence) in which the voltage gradient of glycine and chloride ion is equal to the product of swimming speed, so that these charged particles can At the same speed, there is an obvious boundary between the two ions When glycine and chloride ions enter the concentrated adhesive through the sample interface, there is a low voltage gradient before the moving interface and a high voltage gradient after the interface Because the swimming speed of protein molecules before the moving interface is lower than that of chloride ions, chloride ions can cross rapidly After moving the interface, the protein is in a high voltage gradient, and its swimming speed is faster than glycine Therefore, the moving interface will pile up protein molecules together to form a narrow band The concentration effect of protein in the mobile interface only depends on the concentration of Tris HCl in the sample and concentrated gel (why?) It is independent of the initial concentration of Zui in the sample Because the concentrated glue is macroporous gel, there is no molecular sieve for the protein sample When the mobile interface reaches the interface between concentrated glue and separating glue, the pH of gel increases obviously, leading to a large number of glycine dissociation At this time, the effective swimming rate of glycine increased significantly, surpassing the protein molecules and moving directly after chloride ion As the gel pore size decreases, the migration rate of protein molecules decreases, and the proteins behind it swim in a uniform voltage gradient and pH environment, and are separated according to their intrinsic charge and molecular size 1 What is the effect of gel buffer system on electrophoresis? In SDS-PAGE discontinuous electrophoresis, Tris HCl buffer system is used for gel making buffer, ph6.7 for concentrated gel and ph8.9 for separated gel, while Tris glycine buffer system is used for electrophoresis buffer In the concentrated gel, the pH environment is weak acid, so glycine dissociation is very little, and its swimming efficiency is low under the action of electric field; however, Cl ion is very high, forming a zone with low conductivity between the two, and the protein molecule is in the swimming between the two Because the electric conductivity is inversely proportional to the electric field strength, this region will form a high voltage shaving, and the pressure protein molecules will gather together and concentrate into a narrow region When the sample enters into the separation gel, due to the increase of pH in the gel, it is alkaline, glycine is largely dissociated, and the swimming speed is increased, directly following the chloride ion At the same time, due to the reduction of the pore size of the separation gel, under the effect of electric field, the protein molecules are separated according to their inherent electricity and molecular size Therefore, the influence of pH on the whole reaction system is very important When other factors are excluded from the experiment, the problem can not be solved well, the first consideration should be given to this factor 2 How to deal with samples? According to the different purposes of sample separation, there are three main treatment methods: reduction SDS treatment, non reduction SDS treatment and reduction SDS treatment with alkylation 1) Reduction SDS processing: after adding SDS and DTT (or beta mercaptoethanol) to the buffer, the conformation of the protein is dissociated, and the charge is neutralized to form the molecule combining SDS and protein In electrophoresis, the molecule is separated only according to the molecular weight In general, electrophoresis is treated in this way, the sample is diluted to a proper concentration, added with the sample buffer, centrifuged, boiled for 5min, and then centrifuged 2) Reduction SDS treatment with alkylation: the alkylation of iodoacetamide can protect SH group for a long time and get narrow band; in addition, iodoacetamide can catch excessive DTT and prevent the texture phenomenon during silver staining 10 UL 20% iodoacetamide in 100 UL sample buffer and kept at room temperature for 30 min 3) Non reduced SDS treatment: physiological body fluids, serum, urea and other samples are usually boiled in 1% SDS boiling water for 3min without reductant, so the protein folding is not damaged, so it can not be used as the determination of molecular weight 3 the role of the main components in SDS-PAGE electrophoresis gel? The function of polyacrylamide: acrylamide and providing carrier for protein electrophoresis, its coagulation is directly related to the success of electrophoresis, closely related to coagulant and environment; gel making buffer: ph6.7 for concentrated gel, ph8.9 for separation gel, and Tris HCl system, TEMED and AP: AP provide free radicals, TEMED is a catalyst, which catalyzes the polymerization reaction caused by free radicals; SDS is a cationic detergent, which has four functions: deprotein charge, dissociation of hydrogen bond between proteins, elimination of hydrophobic effect in protein molecules, depeptide folding 4 How to improve the resolution of SDS-PAGE electrophoresis? The full polymerization of polyacrylamide can improve the resolution of the gel Recommended practice: when gelatin is solidified at room temperature, it can be used for a period of time at room temperature Do not use immediately or place in a 4-degree refrigerator The former is easy to cause insufficient solidification, while the latter can cause SDS crystallization General gel can be stored at room temperature for 4 days, and SDS can hydrolyze polyacrylamide Generally, there are three dyes commonly used: amino black, Coomassie brilliant blue and silver Different dyes have different dyeing methods For details, please refer to p82-103 protein electrophoresis technical manual compiled by Guo Yaojun 5 Why does the "smile" (both sides are raised and the middle is concave)? Mainly due to the uneven solidification of the middle part of the gel, most of them appear in thicker gel Treatment method: to be fully solidified before subsequent experiments 6 Why does "frown" (both sides bulge downward and in the middle)? It is mainly found in the protein vertical electrophoresis tank Generally, the air bubbles in the bottom gap between the two plates are not eliminated Treatment method: add a proper amount of buffer between the two plates to eliminate bubbles S1 R) B (| 8 l 7 Why does the belt drag? It is mainly caused by the poor dissolution effect of the sample or the excessive concentration of the separating glue "H" R3 L6} P!} method: centrifugation before adding sample, select appropriate sample buffer, add proper amount of solvent to promote solvent, electrophoresis buffer time is too long, re prepare, reduce gel concentration (8 Why does the texture appear? It is mainly caused by insoluble particles in the sample Treatment method: centrifugation before adding sample; adding proper amount of sample promoting solvent 9 What is "ghost belt" and how to deal with it? "Ghost band" is a kind of protein molecule with complex conformation, which often appears at the top of swimlane (sometimes in concentrated glue) and some unknown bands or sample holes have precipitates at the bottom It is mainly due to the oxidization of reducing agent in the process of heating, which leads to the refolding and re Association of subunits of the original dissociated protein molecules and the polymerization into large ones Molecules, whose molecular weight is larger than the target band, sometimes can not enter the separation gel However, it has the same immunological activity as the target strip It can be seen that it can act as an antibody corresponding to the target strip in WB reaction 8 N / p5d (B, Y9 m, u * | * Z treatment method: after heating and boiling, add appropriate amount of DTT or beta mercaptoethanol to supplement insufficient reducing agent; or add appropriate amount of EDTA to prevent the oxidation of reducing agent 10 Why can't bromophenol blue act as an indicator? In our experiments, we often encounter the phenomenon that bromophenol blue has run out of the bottom of the plate, but the protein has not yet run down It is mainly related to the concentration of buffer and separating glue Treatment method: change the buffer with correct pH value; reduce the concentration of separating glue 1 Why are electrophoresis bands thick? It is common that the bands are very thick in electrophoresis, which is mainly due to the lack of concentration Treatment: increase the length of concentrated gel properly; ensure the pH of concentrated gel storage solution is correct (6.7); reduce the voltage properly; 2 Why is the electrophoresis voltage very high but the current very low? This kind of phenomenon is easy to appear for beginners For example, the voltage is more than 50V, but the current is less than 5mA It is mainly because the electrophoresis tank is not correctly assembled and the current does not form a path Including: a the inner and outer tanks are installed reversely; B the outer tank is too little liquid; C the insulator at the bottom of the electrophoresis tank is not removed (such as the rubber skin for rubber pouring) ）G9 P / P & R "S1 [$U U E treatment method: the electrophoresis tank can be assembled correctly 3 Does the breakage of concentrated glue and separating glue and the bubble between plates affect the electrophoresis? This mainly appears in beginners, generally does not have a great impact on electrophoresis The former is mainly caused by uneven or excessive force of pulling out the comb; the latter is caused by the air entering due to the plate not being pressed tightly after releasing the clamp for glue making "14 gel time is not right, or slow or fast, what's going on? Generally, the glue will set in 30min-1h If the coagulation is too slow, it may be TEMED, APS dose is not enough or failure APS should be used now TEMED is unstable and easy to be oxidized to yellow If the gelation is too fast, it may be that the dosage of APS and TEMED is too much At this time, the glue is too hard and easy to crack, and easy to burn during electrophoresis 15 Is the electrophoresis time longer than normal? It may be due to the PH selection error of the gel buffer system and the electric buffer system, that is, the difference between the PH of the buffer system and the isoelectric point of the separated substance is too small, or the ionic strength of the buffer system is too high.