What is the working principle of laminated glue (or concentrated glue)?
The weak acid in the buffer system, such as glycine, is rarely dissociated at pH 6.7, its effective migration rate is very low, while chloride ion has a high mobility rate, and the protein migration rate is between the two. between. After the voltage is applied, the chloride ion as the lead ion and the glycine ion of the trailing ion are separated, leaving a lower conductivity zone behind it. Since the conductivity is inversely proportional to the electric field strength, this zone obtains a higher voltage gradient, accelerates the migration of glycine ions, catches up with chloride ions, and establishes a voltage gradient of the glycine and chloride ions and the product of the migration rate. The steady state (I don't quite understand this sentence), so that these charged particles move at the same speed, there is a clear boundary between the two ions. When the glycine and chloride interfaces pass through the sample into the concentrated gel, there is a low voltage gradient in front of the moving interface and a high voltage gradient behind the interface. Since the protein molecules move at a lower rate than the chloride ions before the mobile interface, the chloride ions can quickly pass. The protein after moving the interface is in a higher voltage gradient with a faster migration rate than glycine. Therefore, the moving interface stacks protein molecules together to form a narrow zone. The concentration of the protein in the mobile interface depends only on the concentration of the Tris-HCl in the sample and the concentrated gel (why?), regardless of the initial concentration of the zui in the sample. Since the concentrated gel is a macroporous gel, there is no molecular sieve effect on the protein sample. When the moving interface reaches the interface of the concentrated gel and the separating gel, the pH of the gel increases significantly, resulting in a large amount of dissociation of glycine. At this time, the effective migration rate of glycine is significantly increased, surpassing the protein molecule and moving directly after the chloride ion. As the gel pore size becomes smaller, the migration rate of protein molecules decreases, and the proteins that fall behind migrate in a uniform voltage gradient and pH environment, and are separated according to their inherent charge and molecular size.
1. What is the effect of the dispensing buffer system on electrophoresis?
In SDS-PAGE discontinuous electrophoresis, the gelation buffer used was a Tris-HCL buffer system with a concentration of 6.7, a separation gel of pH 8.9, and a Tris-glycine buffer system for the running buffer. In the concentrated gel, the pH environment is weakly acidic, so the dissociation of glycine is very small, and the mobility is low under the action of the electric field; while the CL ion is high, and the band with low conductivity is formed between the two. The protein molecule moves between the two. Since the conductivity is inversely proportional to the electric field strength, this zone forms a higher voltage shaving, and the pressed protein molecules are brought together and concentrated into a narrow zone. When the sample enters the separation gel, it is alkaline due to the increase of pH in the gel, the glycine is largely dissociated, the migration rate increases, directly following the chloride ion, and at the same time, due to the narrowing of the pore size of the separation gel, under the action of the electric field, the protein Molecules are separated according to their inherent chargeability and molecular size.
Therefore, the effect of pH on the entire reaction system is crucial. In the experiment, after the other factors are excluded, the problem cannot be solved well. This factor should be considered first.
2. How is the sample processed?
There are three main treatment methods depending on the purpose of sample separation: reduction SDS treatment, non-reduction SDS treatment, and reduction SDS treatment with alkylation.
1) Reduction SDS treatment: After adding SDS and DTT (or Beta mercaptoethanol) to the loading buffer, the protein conformation is dissociated and the charge is neutralized to form a molecule in which SDS binds to the protein. In electrophoresis, only molecular weight is determined. To separate. Generally, the electrophoresis is treated in this way. The sample is diluted to the appropriate concentration, and the sampled Buffer is added, centrifuged, boiled for 5 minutes, and then centrifuged.
2) Reduction SDS treatment with alkylation: The alkylation of iodoacetic acid amine can protect the SH group well and durable, and obtain a narrow band; another iodoacetic acid amine can capture excess DTT, while preventing texture phenomena when silver is dyed. 10 ul of 20% iodoacetic acid amine in 100 ul of sample buffer and incubated at room temperature for 30 min.
3) Non-reducing SDS treatment: physiological body fluid, serum, urea and other samples are generally only boiled in 1% SDS boiling water for 3 minutes without the addition of reducing agent, so the protein folding is not destroyed and cannot be used as the molecular weight.
3. What is the main component of the SDS-PAGE gel?
The role of polyacrylamide: acrylamide and the carrier for protein electrophoresis, its solidification is directly related to the success or failure of electrophoresis, closely related to the coagulant and the environment;
Gum buffer: The concentration of the gel is pH 6.7, the separation gel is pH 8.9, and the tris-HCL system is selected.
TEMED and AP: AP provides free radicals, TEMED is a catalyst that catalyzes the polymerization caused by free radicals; sodium dodecyl sulfate (SDS): cationic detergent, which has four functions: deproteinization charge, dissociation between proteins The hydrogen bond, the hydrophobic action in the protein molecule is removed, and the polypeptide is folded.
4. Ways to improve the resolution of SDS-PAGE?
The full polymerization of polyacrylamide improves the resolution of the gel. Recommended practice: After the gel has solidified at room temperature, it can be used at room temperature for a period of time. Avoid using the ready-to-use or 4 degree refrigerator. The former is easy to cause insufficient solidification, and the latter can cause SDS to crystallize. Typically the gel can be stored at room temperature for 4 days and the SDS can hydrolyze the polyacrylamide.
Commonly used are amino black, Coomassie blue, silver dyed three dyes, different dyes and different dyeing methods, specifically refer to Guo Yujun's "Protein Electrophoresis Technology Manual" P82-103.
5. “Smile†(both sides are tilted in the middle) What is the reason for the belt?
Mainly due to the uneven solidification of the middle part of the gel, it occurs mostly in thicker gels.
Treatment: After it is fully solidified, it will be used for subsequent experiments.
6. "Frowning" (both sides bulging down) Causes?
It mainly appears in the vertical electrophoresis tank of the protein, and generally the bubble at the bottom gap between the two plates is not excluded.
Treatment: Add an appropriate amount of buffer between the two plates to remove air bubbles. S1 R) b( |8 L
7. Why is there a tailing phenomenon?
Mainly due to poor sample melting effect or excessive concentration of separation gel. " h) R3 l6 |" P! }
Treatment method: centrifugation before sample addition; select appropriate sample buffer, add appropriate amount of sample to promote solvent; electrophoresis buffer time is too long, reconstitute; reduce gel concentration. (
8. Why is there a texture phenomenon?
Mainly caused by sample insoluble particles.
Treatment: Centrifugation before sample addition; add appropriate amount of sample to promote solvent.
9. What is a "ghost belt" and how is it handled?
"Ghost band" is the phenomenon that some macromolecules at the top of the lane (sometimes in concentrated gel) or the bottom of the sample hole are precipitated when running a protein molecule with a complex macromolecular conformation, mainly due to the heating agent. During the process, it is oxidized and loses its activity, causing the dissociated protein molecules to refold and bind and re-associate with the subunits to polymerize into macromolecules, which have a molecular weight larger than the target band and sometimes cannot enter the separation gel. However, it has the same immunological activity in the target strip, and its antibody effect corresponding to the target band can be seen in the WB reaction. 8 N/ p5 D( B, Y9 M, u* |* Z
Treatment method: After heating and boiling, add appropriate amount of DTT or Beta mercaptoethanol to supplement the insufficient reducing agent; or add appropriate amount of EDTA to prevent oxidation of the reducing agent.
10. Why does bromophenol blue not be indicative?
In the experiment, 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. Mainly related to the concentration of buffer and separation gel.
Treatment: Replace the Buffer with the correct pH value; reduce the concentration of the separation gel.
1. Why is the strip of electrophoresis very thick?
It is common for strips to be very thick in electrophoresis, mainly because of the lack of concentration.
Treatment method: appropriately increase the length of the concentrated gel; ensure that the pH of the concentrated gel stock solution is correct (6.7); appropriately reduce the voltage;
2. Why is the electrophoresis voltage high and the current low?
This phenomenon is generally easy for beginners to appear. For example, the voltage is above 50v, but the current is below 5mA. Mainly because the electrophoresis tank is not properly assembled, the current does not form a passage. Including: a. inner and outer tanks are reversed; b. outer tank liquid is too small; c. the insulator at the bottom of the electrophoresis tank is not removed (such as rubber rubber for pouring glue). ) g9 p/ P& r" S1 [$ uu E
Treatment: The electrophoresis tank can be assembled correctly. ,
3. Is the gel and the separation gel broken and there are bubbles between the plates that affect the electrophoresis?
This mainly occurs in beginners and generally does not have much influence on electrophoresis. The main reason of the former is that the pulling of the comb is uneven or excessively strong; the latter is caused by the air entering after the sheet is not pressed after the glue is removed. "
14. The gel time is wrong, or slow or fast, what happened?
Usually the glue is condensed in 30MIN-1H. If the condensation is too slow, it may be TEMED, and the APS dose is insufficient or invalid. APS should be used now, TEMED is unstable and easily oxidized to yellow. If the condensation is too fast, the amount of APS and TEMED may be too much. At this time, the glue is too hard and easy to crack, and it is easy to burn when electrophoresis.
15. Is the electrophoresis time longer than normal?
Possibly due to incorrect pH selection of the gel buffer system and the electrical level buffer system, that is, the difference between the isoelectric point of the pH of the buffer system and the substance to be separated is too small, or the ionic strength of the buffer system is too high.
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