Electrophoresis precast gels

The equipment and reagents for gel electrophoresis are readily available and familiar to laboratory workers. Particularly noteworthy is the steady increase in the popularity of precast polyacrylamide gels since their introduction in the early 1990s (see Section 8.2.4). Precast gels provide researchers with off-the-shelf convenience and reproducibility and help to make gel electrophoresis and IEF commonplace laboratory practices. 

If precast gels are provided, move ahead to the preparation of protein samples for electrophoresis. If gels are to be made, follow these instructions for preparation of an SDS gel of 12% acrylamide. 

Mix components in a 0.5-mL Eppendorf tube and heat in a boiling water bath for 3 minutes to denature the proteins. Cool to room temperature and apply 10 pL of a sample to a well on the gel. Use gel-loading tips on an automatic pipettor to deliver these small samples. One or two wells should contain molecular weight standards. Be sure to record placement of each protein and standards in a sample well in your notebook. Follow the instructions provided with your electrophoresis apparatus and by the supplier of precast gels. 

Numerous protocols for SSCP analysis without the use of radioactive isotopes have been published, and vendors of precast gels and chemicals for preparation of gels offer a detailed description of SSCP electrophoresis. A list of publications and Web sites is given in Table 5.1. Commercially available precast gels may help to ensure the reproducibility of results between laboratories. The reliability of SSCP performed... 

FIGURE 10.10 (a) coli proteins separated by isoelectric focusing in a pH 4-6 precast gel in the first dimension and by electrophoresis in an SDS/8-l8% polyacrylamide gel in the second dimension, blotted onto a PVDF membrane and stained with Coomassi brilliant blue, (b) MALDI-TOF mass spectrum of in situ tryptic digest of spot I, identified as cysteine synthase A. Reprinted with permission from reference 16. 

Figure 15.5 IEF gel electrophoresis of formalin-treated RNase A (a) and its fractions (b) separated by gel filtration, (a) M, IEF markers (pi) lane 1, unfractionated formalin-treated RNase A. (b) lane 1, monomer lane 2, dimer lane 3, trimer lane 4, tetramer lane 5, pentamer. IEF gel electrophoresis was performed using precast 5% polyacrylamide gels with a pi range of 3-10. The pi range for the individual oligomers in 15.5b appears greater than that for the unfractionated sample because a higher concentration of protein was used for the lanes in 15.5b. This results in an increased staining intensity for the oligomers at the pi extremes, which are minor components of the unfractionated sample in 15.5a. See Rait et al.10 for details.

Obtain a precast SDS-polyacrylamide slab gel or prepare one according to instructions in Experiment 4. The recommended gel is 12°/o acrylamide with a thickness of 0.75 mm. Protein samples are prepared as follows Purified proteins (transferrin, bovine serum albumin, a, -antitrypsin, a-lactalbumin from Experiment 4, and molecular weight standards) are supplied in Tris buffer, pH 6.8 solutions at a concentration of 1 mg/mL. Sera samples have been diluted and are ready for use. Prepare protein samples for electrophoresis in 0.5-mL microcentrifuge tubes with attached caps. Label the tubes from 1 to 5 as below or per your Instructor s directions. 

Approximately 2 hours are required to prepare and incubate the restriction enzyme reaction mixtures. The agarose slab gel may be prepared during the incubation period. Up to 1 to 2 hours are required to prepare the gel slab. Alternatively, precast agarose gels may be provided. The electrophoresis may be completed as a group project. Most electrophoresis chambers accommodate slab gels with 15 to 20 sample wells. 

A variety of precast IPG gels is commercially available from electrophoresis suppliers, and this represents another significant advantage over carrier ampholyte... 

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