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SECOND DIMENSION FORMATS
The second dimension of a 2D gel separation is used to separate proteins by molecular weight. At the CNRU Mass Spectrometry Core Facility we have available a wide variety of gel sizes for your separation needs. The table below provides some examples of things to keep in mind when deciding upon a gel format. These gels are precast and are commercially available. 2D
| FORMAT | APPLICATION | CHALLENGE |
| 7 cm | Rapid screening of co-immunoprecipitated samples. | Searching for changes of a few proteins in a specific molecular weight and pI range, where only a few samples are to be run and the results are needed as early as possible. |
| 11 cm | Separation of protein interaction complexes or immunoprecipitation experiments, where there are hundreds of proteins and a large number of samples to process. | Analysis of changes in protein patterns with a subset of proteins in the sample and many samples to run. |
| 17, 18, 24 cm | A screening project to identify statistically significant changes in protein patterns among many patient samples and controls over a time course of disease progression. | Monitoring protein expression changes for all proteins in a project with complex samples and many gels to run. For low fluorescence applications such as DIGE, we need to cast our own gels. The tables below contain recipes we use for 10% and 12.5% gels. |
10% Polyacrylamide
| Vendor and # | | 10 mL | 50mL | 100mL | 150 mL |
| | Nanopure H2O | 4.07 mL | 21 mL | 42 mL | 62.5 mL |
| BioRad 161-0798 | 1.5 M Tris HCl pH 8.8 | 2.5 mL | 12.5 mL | 25 mL | 37.5 mL |
| BioRad 161-0418 | 20% SDS | 50 無 | 0.25 mL | 0.5 mL | 0.75 mL |
| BioRad 161-0158 | 30% Acrylamide | 3.33 mL | 16.7 mL | 33 mL | 50 mL |
| BioRad -0700 | 10% APS | 50 無 | 250 無 | 500 無 | 750 無 |
| BioRad 161-0800 | TEMED | 4.5 無 | 22.5 無 | 45 無 | 67.5 無 |
| | | Small gel | 1 gel | 2 gels | 4 gels |
12.5% Polyacrylamide
| Vendor and # | | 10 mL | 50mL | 100mL | 150 mL |
| | Nanopure H2O | 3.35 mL | 16.75 mL | 33.5 mL | 50 mL |
| BioRad 161-0798 | 1.5 M Tris HCl pH 8.8 | 2.5 mL | 12.5 mL | 25 mL | 37.5 mL |
| BioRad 161-0418 | 20% SDS | 50 無 | 0.25 mL | 0.5 mL | 0.75 mL |
| BioRad 161-0158 | 30% Acrylamide | 4 mL | 20 mL | 40 mL | 60 mL |
| BioRad 161-0700 | 10% APS | 50 無 | 250 無 | 500 無 | 750 無 |
| BioRad 161-0800 | TEMED | 4.5 無 | 22.5 | 45 無 | 67.5 無 |
| | | Small gel | 1 gel | 2 gels | 4 gels |
Look here for recipes for running buffers, sealing agarose and ampholytes.
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