What is the effect of adding Tris HCL to the separating glue?

When formulating the separating gel, if Tris-HCl is added, it may have the following effects on the running gel:

Buffering capacity: Tris-HCl is a buffer whose purpose is to maintain pH stability. The addition of Tris-HCl increases the buffering capacity and helps to maintain pH stability, which may be beneficial for some experiments.

However, too much Tris-HCl may affect the results of some experiments, such as the stability or activity of the protein.

Ionic strength: The addition of Tris-HCl may increase the ionic strength of the solution, which may affect the efficiency of electrophoresis. Solutions with high ionic strength may reduce the speed at which ions move, affecting the effectiveness of electrophoresis.

Protein stability: The pH of Tris-HCl may affect the stability of the protein. A pH that is too high may make the protein unstable, which can affect the effectiveness of electrophoresis.

Electrode protection: Tris-HCl is a weakly alkaline substance that protects the electrodes from corrosion. However, too much Tris-HCl may affect the function of the electrode and thus the effectiveness of electrophoresis.

Therefore, when preparing the separating gel, it should be prepared in strict accordance with the formula and avoid adding too much Tris-HCl. If additional Tris-HCl is added, the gel can be reconstituted to ensure the accuracy of the experimental results.

In SDS-PAGE discontinuous electrophoresis, gel preparation buffer.

The Tris-HCl buffer system is used, and the stacking gel is, separating gel; Whereas the electrophoresis buffer uses tris-glycine.

Buffer system. In the stacking gel, its pH environment is weakly acidic, so there is little dissociation of glycine, and its swimming efficiency is low under the action of the electric field. On the other hand, the Cl ion is very high, and a less conductive band is formed between the two, and the protein molecule swims between the two. Due to the conductivity and electric field strength.

Inversely proportional, this zone forms a higher voltage shaving, and the pressed protein molecules come together and condense into a narrow band. When the sample enters the separating gel, due to the increase of pH in the gel, it is alkaline, and glycine is dissociated in large quantities, and the swimming rate increases, directly following the chloride ion.

Then, due to the shrinking pore size of the separating gel, the protein molecules are separated according to their intrinsic chargeability and molecular size under the action of an electric field.

The function of Tris-HCl in the stacking gel in DS electrophoresis is to increase the ionization rate.

The concentration of protein at the mobile interface only depends on the concentration of Tris-HCl in Yana fruit stacking gel, and there is no zeolite effect because the stacking gel is a large-pore diameter gel.

However, when the mobile interface reaches the interface of the concentrated gel separation, the pH of the gel increases significantly, resulting in a large number of dissociation of GLY, an increase in the effective swimming rate, and a movement beyond the protein after chloride ions. At the same time, the pore size of the gel decreases, so that the protein mobility decreases. The protein molecules then swim in a voltage gradient and are finally separated according to their chargeability and molecular size.

I have done it once before that the marker and the sample are only separated in the upper part of the separating gel, and the lower half is not separated at all, and the marker strip is not visible at all.

15% glue should be fine, run longer, longer distance, you can run away. The amount of sample loading depends on the expression of your protein, generally 15-30 g should be no problem, but if -actin is used as a control, it is recommended to use another 12% gel to run -actin, so that the width of the target protein and the control band are about the same.

You can try small molecule glue, but 15% is no problem. The amount of sample loaded is not very important, depending on the concentration of your sample loaded.

Can't run away? Test dyeing? If it's a miscellaneous antibody, there's no problem of not being able to run away, right? Or run with a big glue if anybody of you guys can get it.

There will be an impact.

There is less separating gel and more stacking gel, which means that the protein separation process will be shortened. Although the glue concentration will not change, the proportion of glue will be smaller than that of normal glue. It's like the feeling of a normal screen changing to a widescreen.

The choice of gel concentration in Western blot depends on the molecular weight of the protein of interest, different gel concentrations have different resolutions for different molecular weights of the target protein, for example, 15% gel is more suitable for 45 kDa protein, and 10% gel is more suitable for 70 kDa protein of interest, and because the membrane of Western blot should be a gel that completely covers the SDS Page, there is no need to worry too much about the bands on the gel Therefore, the choice of gel concentration in Western blot depends on the molecular weight of the protein of interest.

If the bromophenol blue runs to the bottom of the glue, 26kd is 10% from the bottom to the top of 1 4, and 15% is from the bottom to the top of about 1 2 (not very sure), for the protein with a relatively small molecular weight, of course, the greater the concentration of the gel, the better the separation effect. But you have to take into account that once the pore size of the gel is smaller than the diameter of the protein-SDS complex, all proteins with molecular weights greater than this protein still do not have good separation. If the pore size is too large, all the proteins run so fast that they can't be separated.

Therefore, the key is to look at the protein near this molecular weight, such as all proteins within 5kd, the velocity difference in different concentrations of glue should be as large as possible, assuming a lognormal distribution, then the variance should be large enough to have a better separation effect. On the other hand, the greater the distance the egg whites run, the more they can separate themselves from nearby proteins.

My suggestion is that bromophenol blue runs to the end, and the target protein is exactly 1 to 2 places of the whole gel, and the effect is best when isolated.

My understanding is to maintain a certain concentration of SDS throughout the electrophoresis system to ensure that the protein is fully bound to SDS, otherwise it may affect the electrophoresis effect.

Biochemical experiment preview thinking questions. Unfortunately, there is no answer.