In SDS-PAGE discontinuous electrophoresis, Tris-HCL buffer system was used to prepare gel buffer, and the pH of concentrated gel was 6.7 and that of separated gel was 8.9. Tris-glycine buffer system is used for electrophoresis buffer. In concentrated gel, the pH environment is weakly acidic, so glycine is rarely dissociated, and its swimming efficiency is low under the action of electric field. However, the CL ion is very high, and a region with low conductivity is formed between them, and protein molecules wander between them. Because the conductivity is inversely proportional to the electric field strength, a high voltage gradient is formed in this region, which forces protein molecules to gather together and concentrate in a narrow region. When the sample enters the separation gel, due to the increase of pH value in the gel, it is alkaline, and glycine is dissociated in large quantities, and the swimming rate is increased, which is directly behind chloride ion. At the same time, due to the decrease of the pore size of the separation gel, protein molecules are separated according to their inherent electrification and molecular size under the action of electric field.
Therefore, the influence of pH value on the whole reaction system is very important. If the problem cannot be solved well after excluding other factors in the experiment, this factor should be considered first. Of course, other factors can also be considered from many aspects.
How to deal with the sample?
According to the different purposes of sample separation, there are three main treatment methods: reduced SDS treatment, non-reduced SDS treatment and reduced SDS plus alkylation treatment.
1) Reduction SDS treatment: After adding SDS and DTT (or β-mercaptoethanol) into the loading buffer, the protein conformation dissociates, and the charge neutralizes, forming a molecule that binds SDS to protein, and the electrophoresis is only separated by molecular weight. Generally, electrophoresis is treated in this way. Dilute the sample to an appropriate concentration, add the loading buffer, centrifuge, boil in boiling water for 5 minutes, and then centrifuge to add the sample.
2) Alkylation-reduction SDS treatment: Alkylation of ammonium iodoacetate can protect SH group well and firmly for a long time, and obtain a narrow band; In addition, amine iodoacetate can capture excessive DTT and prevent the texture phenomenon in the process of silver staining. 100ul sample buffer contains 10ul 20% amine iodoacetate, and it is kept at room temperature for 30 minutes.
3) Non-reducing 3-min SDS treatment: physiological body fluid, serum, urea and other samples are usually boiled in boiling water of 1%SDS for 3 minutes without adding reducing agent, so the protein fold is not destroyed and cannot be used for molecular weight determination.
3. What are the functions of the main components in 3.SDS-PAGE gel?
The function of polyacrylamide: acrylamide provides a carrier for protein electrophoresis, and its curing is directly related to the success of electrophoresis and closely related to coagulant and environment;
Gel-forming buffer: pH6.8 is selected for concentrated gel and pH8.8 for separated gel. Tris-HCl system, TEMED and AP: AP catalyst were selected to catalyze the polymerization of monopropene and dipropene to produce polyacrylamide. Tetramethyl ethylenediamine; Coagulant; Accelerating AP catalysis; Sodium dodecyl sulfate (SDS), an anionic detergent, has four functions: removing protein charge, dissociating hydrogen bond between protein, removing hydrophobic interaction in protein molecule and removing polypeptide folding.
How to improve the resolution of SDS-PAGE electrophoresis?
Sufficient polymerization of polyacrylamide can improve the resolution of gel. Suggested practice: After the gel is solidified at room temperature, it can be used at room temperature for a period of time. Avoid ready-to-use or 4-degree refrigerator, the former may lead to insufficient coagulation, and the latter may lead to SDS crystallization. Generally, gel can be stored for 4 days at room temperature, and SDS can hydrolyze polyacrylamide.
There are three commonly used dyes: amino black, coomassie brilliant blue and silver dyeing, and different dyes have their own different dyeing methods. For details, please refer to P82- 103, Handbook of Electrophoresis Technology in protein, edited by Guo.
5. What is the reason for the "smile" (the two sides are upturned and the middle is concave)?
Mainly due to the uneven curing of the middle part of the gel, it often appears in thicker gel.
Treatment method: Follow-up experiments shall be conducted after it is completely cured.
[6] What is the reason for the "frown" ribbon?
It mainly appears in the vertical electrophoresis tank in protein, and the bubbles in the gap between the bottom of the two plates are not completely removed.
Solution: A proper amount of buffer solution can be added between the two plates to eliminate bubbles.
⒎ Why does the belt tail?
It is mainly caused by the poor melting effect of the sample or the excessive concentration of separation glue. In addition, it may be that there is too much separation glue when pouring glue, resulting in too little concentrated glue, which can not concentrate and does not press the sample into a line.
Treatment method: centrifuge before adding sample; Select an appropriate sample buffer and add an appropriate amount of sample accelerator; The electrophoresis buffer is too long and needs to be reconstituted; Reduce the gel concentration; Don't divide too much glue when pouring it.
⒏ Why does the band have texture phenomenon?
It is mainly caused by insoluble particles in the sample.
Treatment method: centrifuge before adding sample; Add an appropriate amount of sample promoting solvent.
What is the "ghost belt" and how to deal with it?
"ghost area" means that some unknown macromolecular bands often appear at the top of the lane (sometimes in concentrated gel) or precipitate at the bottom of the loading hole when running protein molecules with complex macromolecular conformation. The main reason is that the reducing agent is oxidized and loses its activity during the heating process, which makes the previously dissociated protein molecules refold and combine, re-associate with subunits, and polymerize into macromolecules with molecular weight greater than the target band, sometimes unable to enter the separation gel. But it has the same immune activity in the target strip and can react with the antibody corresponding to the target strip in WB reaction.
Treatment: after heating and boiling, add a proper amount of reducing agent which is insufficient to be supplemented by DTT or β mercaptoethanol; Or an appropriate amount of EDTA can be added to prevent oxidation of the reducing agent.
⒑ Why can't bromophenol blue play an indicating role?
In the experiment, we often encounter the phenomenon that bromophenol blue has run out of the bottom of the board, but protein has not. It is mainly related to the concentration of buffer and separation gel.
Solution: Replace the buffer; Have the correct pH value; Reduce the concentration of separating glue.
(1) Why is the electrophoretic band thick?
It is very common to have thick bands in electrophoresis, mainly because of the concentration difference.
Solution: appropriately increase the length of concentrated glue; Ensure that the pH value of the concentrated glue storage solution is correct (6.7); Reduce the voltage appropriately;
5] Why is the electrophoresis voltage high and the current low?
This phenomenon is very common for beginners. For example, the voltage is above 50v, but the current is below 5mA. The main reason is that the electrophoresis tank is improperly assembled and the current does not form a path. Comprises the following steps: a, reversely installing an inner groove and an outer groove; B. There is too little liquid in the outer tank; C. The insulator at the bottom of the electrophoresis tank has not been removed (for example, rubber skin for glue filling).
Solution: The electrophoresis tank can be assembled correctly.
⒔ Separation gel and concentrated gel are broken, and there are bubbles between the plates, which will affect electrophoresis?
This mainly appears among beginners, and generally has little effect on electrophoresis. The former is mainly caused by uneven or excessive force when pulling the comb; The latter is caused by the fact that after the rubber-making fixture is loosened, the plate is not pressed tightly and air enters.
5. The gel time is wrong, slow or fast. What happened?
Usually, the glue will solidify within 30 minutes-1h. If coagulation is too slow, it may be TEMED and APS dose is insufficient or ineffective. APS should be ready to use. TEMED is unstable and easily oxidized to yellow. If coagulation is too fast, APS and TEMED may be used too much. At this time, the glue is too hard and easy to crack, and it is easy to burn during electrophoresis.
5] The electrophoresis time is longer than normal?
It may be due to the wrong choice of PH value of gel buffer system and electric buffer system, that is, the difference between PH value of buffer system and isoelectric point of separated substance is too small, or the ionic strength of buffer system is too high.
[6] Why do you need to add water immediately after adding the separation glue?
Immediately after adding the separation gel, cover it with a layer of double distilled water. First, in order to keep the interface level of the separation gel, water can flatten it and make protein molecules run on the same horizontal plane; The second is to prevent the inhibition of gel polymerization by oxygen in the air.
⒘ Preparation of continuous separation gel system (gel plate thickness 0.75mm, length 10cm) 100ml system: double distilled water 37.5ml.
Urine 20-50g
10×TBE buffer 8 ml.
30% acrylamide10ml
APS (ammonium persulfate) 500-800 ul[ Note: available at any time]
TEMED (tetramethylethylenediamine) 23.5 microliters