Buffers electrophoresis

Prepare 100 mL (500 mL if the gel is to be run under the buffer) of an electrophoresis buffer that is 20 mM tris-(hydroxymethyl)amino methane (TRIS or THAM), 6 mM sodium acetate, and 1 mM disodium EDTA. Adjust the pH of this solution to 7.9 using concentrated HC1. Also prepare small volumes of solutions of hemoglobin and cytochrome C in the buffer (the concentration is not important) and also a mixture solution of these two solutes. Add a quantity of sucrose to each. 

What are some requirements for an effective electrophoresis buffer ... 

An electrophoresis buffer must have ions to carry current, and must not interfere with the stability of the sample. 

Almost any buffer can be used for electrophoresis in a continuous system. Solutions of relatively low ionic strength are best suited for electrophoresis because these keep heat generation at a minimum. On the other hand, protein aggregation may occur if the ionic strength is too low. The choice of buffer will depend on the proteins being studied, but in general the concentrations of electrophoresis buffers are in the range of 0.01 to 0.1 M. 

Tris-sulfate/Tris-borate, Tris-formate/Tris-borate, and Tris-citrate/Tris-borate have been advocated as electrophoresis buffers.5 For basic proteins, a low-pH alanine-acetate system28 is often used. 

Transfer tanks are made of plastic with two electrodes mounted near opposing tank walls. A nonconductive cassette holds the membrane in close contact with the gel. The cassette assembly is placed vertically into the tank parallel to the electrodes and submerged in electrophoresis buffer. A large volume of buffer in the tank dissipates the heat generated during the transfer. 

Fig. 5 Screening by affinity CE for interaction of SAP peptides with heparin in solution. An endoproteinase Asp-N-treated Glu-C digest of SAP solubilized in water was injected for 12 s and subjected to CE at 15 kV (detection at 200 nm) in the presence of heparin (Hep) (B) added to the electrophoresis buffer (0.1 M phosphate, pH 7.5) at the concentration indicated. The peptide marked with asterisks was identified by spiking with HPLC-purified fragments and corresponds to the fragment in Figure 6. (From Ref. 71.)...

Fig. 3 ACE/MS of a synthetic all-D, Fmoc-DDXX library of 100 tetrapeptides using vancomycin as the receptor. (A-D) Selected ion electropherograms for the masses indicated (E) reconstructed ion electropherograms for runs without (left) and with (right) vancomycin in the electrophoresis buffer. See text for conditions and further explanations. (Reprinted with permission from Ref. 38. Copyright 1995 American Chemical Society.)...

Add the samples with a syringe (e.g., Hamilton syringe) or special pipette tips to form a lower layer below the electrophoresis buffer within the slots. 

Sometimes the electrophoresis buffer produces a high background. A significant, rapid increase of electric field strength, as... 

Two of 4.0 mL of electrophoresis buffers are introduced into the reservoirs of SDS-PAGE part. 

The part is positioned at the tapered surface of PAG on the SDS-PAGE part containing 4.0 mL of each electrophoresis buffer (see Note 6). 

The surface of the IPG should be buried into the tapered gel by adjusting 0.5 mm downward from the surface of the tapered gel when the IPG is attached on the surface. Then, the proteins are transferred into the tapered gel without leaking to electrophoresis buffer. 

Submarine gel chamber with an external circulation through heat exchangers to cool the electrophoresis buffer. 

Standard research-grade agarose is usually sufficient, but special agaroses may be used for specific applications (e.g. high-resolution gels). The electrophoresis buffer is usually prepared and stored as a 10 x concentrated stock. The most commonly used buffer is Tris-borate-EDTA (TBE), or alternatively, one may use Tris-acetate-EDTA (TAE) buffer ... 

Electrophoresis buffer Tris, glycine, SDS, pH 8.2 2-Mercaptoethanol (STENCH )... 

Electrophoresis buffer, Tris-acetate buffer listed above containing ethidium bromide, 0.5 p,g/mL... 

Add electrophoresis buffer to the reservoirs and connect the power supply. 

---