Stacking gel

The IEF mixture is poured between the gel plates up to the top and the comb is inserted (no stacking gel is required). Once the gel has set, the comb is removed and the wells are rinsed with distilled water. Any potential leaking points of the gel apparatus need to be sealed with 2% agarose as communication between the upper and lower chambers, must be avoided. 

Till thattime, make the stacking gel mix excluding APS and TEMED. 

Prepare resolving gel mixture and stacking gel mixture as showed in Table 1. 

Add the stacking gel mixture on top and insert the gel comb. Wait for another 30 min. After the stacking gel is set, remove the comb and put the gel with the stand into the running tank. 

The components of the resolving gel and stacking gel for separation of proteins with different molecular weights... 

Place test and reference solutions, contained in covered test-tubes, in a waterbath for 2 min. Apply 10 ul of reference solution (f) and 50 pX of each of the other solutions to the stacking gel wells. Perform the electrophoresis under the conditions recommended by the manufacturer of the equipment. Detect proteins in the gel by silver staining. 

Addition of 0.01% bromophenol blue to buffer C makes it easier to recognize of the slots of the stacking gel and also allows the identification of the electrophoresis front during run. 

The stacking gel is made just before performing the electrophoresis. The residual liquid on top on the separation gel is removed completely using small pieces of filter paper. Then the stacking gel mixture is added and the slot former ( comb ) is in-... 

After polymerization, just before performing the electrophoresis, the stacking gel is prepared as described previously. 

Start the electrophoresis immediately when all samples, marker protein mixtures, or references are applied, because molecules diffuse through the soft stacking gel and the pH jump between stacking gel and separation gel, which is important for separation power, drops down. 

As a general rule, a voltage of 30-40 volts is useful for introducing the sample into the stacking gel and field strength of 10-15 V/cm separation gel length for the separation is sufficient. If an efficient cooled device is used, the field strength maybe increased. 

Since a stacking gel is not necessary, put the comb directly into... 

Prepare the separation gel and the stacking gel according to Table 2.6. The separation gel may be poured separately and overlaid with ddH20, but the stacking gel also can be carefully poured directly onto the fresh (liquid) separation gel. 

Mix the gel according to Table 2.7, pour the mixture into the cassette and cover with Soln. 1 (Table 2.7). The polymerization takes place very slowly therefore, the gel should be prepared 36-48 h before electrophoresis. A stacking gel is not needed. 

Solutions are gently mixed according to Table 2.9 in the indicated order and filled up with ddH20. Start the polymerization by addition of Soln. F and pour the mixture immediately into the gel cassette. When the appropriate height is reached, cover the liquid with water or n-butanol to get a smooth surface. Prepare the stacking gel as short as possible before starting the electrophoresis to avoid a decrease of the pH jump between stacking and separation gel by diffusion. 

Prepare the gel as described in Protocol 2.1.1, i.e., first a separation gel followed by a stacking gel without sample application slots. 

Place the IPG strip face down onto the stacking gel, about 5 mM apart of the cathode paper bridge. Place silicone rubber sample applicators for molar mass marker proteins at one or both sides of the IPG strip. 

Monomer solutions for resolving gel and stacking gel are poured in the cavity and polymerized. To minimize the entire size of the part, both the gel dimension and buffer amount are reduced. Rapid separation is performed without an increase... 

SDS-polyacrylamide gel electrophoresis Protein samples were analyzed under denaturing conditions in a discontinuous gel system as described by Laemmli [13] using a 5% stacking gel and a 12% separating gel in a vertical gel system (BioRad, Mini Protean 11, CA, USA). 

In contrast to horizontal systems, it is not essential to use stacking gels in a vertical system. In such a system, protein zones in the strips are concentrated and IEF gel with low polyacrylamide concentration behaves like a stacking gel. 

The process of disc gel electrophoresis. A Before electrophoresis. B Movement of chloride, glycinate, and protein through the stacking gel. 

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