SDS - Page electrophoresis

The SDS - Page electrophoresis mainly USES the molecular sieve effect of polyacrylamide to isolate proteins and nucleic acids, and the SDS - PAG protein electrophoresis analysis can isolate the protein from the size of the protein subunit.

Almost all proteins are conducted on polyacrylamide gel electrophoresis analysis, and always want to make sure that all the conditions from protein solution into a single polypeptide subunits in hand are likely to reduce the mutual gathered themselves together, and the most commonly used is the sds-page electrophoresis, about everyone some problems often encountered in the process of discussion:

Q: the basic principle of SDS - PAGE electrophoresis?
A: SDS polyacrylamide gel electrophoresis, is introduced in polyacrylamide gel system SDS (sodium dodecyl sulfate), SDS and degeneration of polypeptide, protein and negatively charged, because the peptide in combination with the amount of SDS almost always proportional to the molecular weight of peptides and had nothing to do with, so the SDS peptide compounds in propylene amide gel electrophoresis migration rate is only related to the size of the peptides, in saturated state, the peptide can be combined with 1.4 g detergent per gram.
When the molecular weight between 15 kd and 200 kd, the migration rate and molecular weight of protein logarithm linear relationship, conform to the type: logMW = K - bX, type: MW as molecular weight, X for mobility, K and b are constant, if the mobility of standards of known molecular weight protein on molecular weight of logarithmic mapping, a standard curve can be obtained, and the unknown protein electrophoresis under the same conditions, according to its electrophoretic mobility of molecular weight on the standard curve can be obtained.

Q: the effect of adhesive buffer system on electrophoresis?
A: in the SDS - page discontinuous electrophoresis, the rubber buffer is used in the Tris - HCL buffer system, which is pH6.7, and the separation gel is pH8.9;
The Tris - glycine buffer system used in electrophoresis buffer.
In concentrated glue, the pH environment is weakly acidic, so the glycine solution is very small, and its effect is low.
But the CL ion is very high, and the low conductivity between the two, the protein molecules are in between.
Since the conductivity is inversely proportional to the electric field intensity, the zone has formed a high voltage shaves, and the protein molecules are gathered together to condense into a narrow band.
After the sample into the separation of glue, due to the increase of pH in the glue, alkaline, glycine dissociation in great quantities, the rate of migration increased, directly after the chloride ion, at the same time due to the separation gel pore size reduction, under the action of electric field, the protein molecules according to its inherent charged and molecular size separation.
Therefore, the effect of pH on the whole reaction system is very important. In the experiment, it is not easy to solve the problem after excluding other factors, and the factor should be considered in the first place.

Q: how do you handle the sample?
A: according to the separation of samples, there are three main methods: reduction of SDS treatment, non-reduction SDS treatment, and the reduction of SDS with alkylation.
1, the reduction of SDS processing: add SDS in the sample buffer and DTT (or Beta mercaptoethanol), protein conformation by dissociation, charge neutralization, form of SDS combined with protein molecules, in electrophoresis, only separated according to molecular weight.
The normal electrophoresis is processed in this manner, the sample is diluted with appropriate concentration, the sample is added to the sample Buffer, centrifuge, boiling water is boiled 5min, and then the centrifuge is added.
2. Reduction SDS with alkylation action: alkylation of iodoacetic acid can be a good and durable and strong protective SH group, with a narrow band;
Other iodoacetic acid amine can trap excessive DTT, and prevent the texture phenomenon of silver dye.
100 mu l sample buffer of 10 mu l 20% iodoacetic acid, and at room temperature for 30min.
3, the reduction of SDS processing: physiological fluid, serum and urea samples, generally only 1% SDS boiling water 3 min, without reductant, and protein folding is not damaged, cannot be used as the determination of molecular weight.

Q: the main components of the SDS - PAGE electrophoresis gel?
A: the role of polyacrylamide: acrylamide and the carrier for protein electrophoresis, its solidification is directly related to the success of electrophoresis, which is closely related to the coagulant and environment;
Glue buffer solution: concentrate the choice of pH6.7, separation of glue choice pH8.9, select tris - HCL system, the specific role of the introduction;
TEMED and AP: coagulation, accelerating the coagulation of polyacrylamide;
Sodium dodecyl sulfate (SDS) : cationic detergent, which ACTS on four: to remove the hydrogen bond between proteins, to remove the hydrophobic effect of protein molecules and to fold the polypeptide.

Q: how to improve the resolution of SDS - PAGE?
A: the full polymerization of polyacrylamide can improve the resolution of gels.
Recommended practice: after the gel is set at room temperature, it can be used at room temperature for a period of time.
Avoid to be with the use or 4 degree refrigerator to place, former easy to cause solidification not sufficient, the latter can cause SDS crystallization.
Gels can be stored at room temperature for 4 days and SDS hydrolyzed polyacrylamide.
2. Generally used, there are three dyes, such as amino black, coomassie bright blue and silver, and different dyeing methods of different dyes.

Q: what is the reason for the "smile"?
A: mainly due to the uneven solidification of the middle part of the gel, it appears in the thicker gel.
Treatment: to be fully solidified for subsequent experiments.

Q: what is the reason for "frowning" (bulging on both sides)?
A: it mainly appears in the protein vertical electrophoresis tank, usually between the two sides of the bottom gap bubble.
Treatment: add a moderate amount of buffer between the plates to remove the bubbles.

Q: why is there a tow - tailed phenomenon?
A: it is mainly caused by the dissolving effect of the sample and the excessive concentration of the separation gel.
Treatment: pre-sample centrifugation;
Select the appropriate sample buffer and add the appropriate sample to promote the solvent;
The electrophoresis buffer is too long and reformulated;
Reduce the gel concentration.

Q: why does it occur?
A: mainly caused by the insoluble particles of the sample.
Treatment: pre-sample centrifugation;
Add a proper amount of sample to promote the solvent.

Q: what is the "ghost belt"? How do you handle it?
A: "ghost" is running in macromolecular conformation of complex protein molecules, often appear at the top of the lane (sometimes) in the concentrated glue of some macromolecules unknown sample stripe or add holes at the bottom of the sediment is mainly due to the reducing agent in the process of heating was oxidized and lose activity, led by original solution from the protein molecules to fold and sub association, aggregation into large molecules, its molecular weight than target stripe, sometimes can't into the separation of glue.
However, it has the same immunological activity in the target bar, and in the WB reaction, it can be seen that it can be related to the target band.
Treatment: after heating and boiling, add a moderate amount of DTT or Beta mercaptoethanol to supplement the deficient reducing agent;
Or an amount of EDTA can be added to prevent the reagent oxidation.

Q: why is bromophenol blue not indicative?
A: we often encounter bromophenol blue at the bottom of the board in the experiment, but the protein hasn't come off yet.
It is mainly related to the concentration of buffer and separation gel.
Treatment: replace Buffer with correct pH value;
Reduce the concentration of the separation gel.

Q: why is the ribbon of the electrophoresis very thick?
A: the ribbon in the electrophoresis is very common, mainly because it is not concentrated.
Treatment: increase the length of the concentrate properly;
Ensure the correct pH (6.7) of the concentrated liquid.
Reduce voltage properly;

Q: why is the electrophoretic voltage high and the current low?
A: this phenomenon is easy to come by.
For example, the voltage is over 50v, but the current is below 5mA.
The main result is that the electrophoresis tank is not properly assembled and the current is not formed.
Including: a. Inside and outside tank;
B. Too little fluid in the outer tank;
C. The insulator at the bottom of the electrophoresis groove has not been removed (such as rubber covering rubber).
Treatment: the electrophoresis groove is correctly assembled.

Q: is there any influence on the electrophoresis between the cracking of the concentrated glue and the separation glue and the air bubbles between the plates?
A: this is mainly for beginners. It doesn't have much effect on electrophoresis.
The former is mainly due to unevenly or overexertion of plucking comb;
The latter is caused by the uncompressed air entering the plate after the glue is removed.

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