Before running protein samples using gel electrophoresis, it is useful to know the anticipated position of the bands on the gel by calculating the anticipated size of the protein. Chapter 6 of Voet and Voet outlines basic principles of SDS-PAGE. Knowing the anticipated size of the bands is also important when selecting the concentration of polyacrylamide that the gel should contain, since different levels of polyacrylamide are more efficient at separating different ranges of protein sizes. The sources Molecular Cloning (which also discusses the stain used in this process, Coomassie G-250) and Bio-Rad manual are also good resources for principles of gel electrophoresis.
Deep Thoughts on Controls
What is the purpose of a control? Generally, a control can be used to verify that your system is behaving how you would expect it to. Additionally, a control can be used to track down what is happening if/when something goes wrong in your experiment and correct it. In this laboratory, you will be required to use controls in at least two contexts: protein expression (for ncAA incorporation) and assays. Are there any other controls you should be incorporating into your experiments?
For your protein expression, you should be setting up both a positive and negative controls. What is each of these controls? What results do you expect to observe from your positive and negative controls? If your controls deviate from this expectation, what is going on? Do you need any purification controls?
For your assay, a control is an important way to verify that the observed activity is coming from your protein and not from another source. How should this control be set up? It’s okay if some activity is observed in your control- this is often referred to as “background.” In this case, how might you use the results of this control?
Necessary Materials for SDS-PAGE
- Glass plates, casting stand, casting clamps, comb
- Gel Rigs (with dams)
- Gel-loading tips
- Bottles for 1X Running Buffer,
- Plastic gel storage containers (one per group)
- pH meters-common stations.
SDS-PAGE-refer to Bio-Rad SDS-PAGE guide for Mini Protean II System discontinuous Laemmli gels
- 30% polyacrylamide solution (Caution: neurotoxin—wear gloves, goggles, lab coat) (aliquots provided)
- Temed (purchased as solution)
- Ammonium persulfate-10% wt/vol in water; make fresh daily (1 mL)
- Separation gel buffer (1.5 M Tris pH 8.8) (make 100 mL)
- Stacking gel buffer (0.5 M Tris pH 6.8) (make 100 mL)
- 10% SDS solution (prepared for you )
- 2X SDS load dye, pH 6.8 (contains 125 mM Tris-HCl, 4% SDS, 20% glycerol, 0.02% bromophenol blue, and 5% b-mercaptoethanol) (10 mL)
- 10X Running Buffer, pH 8.3 (contains 250 mM Tris base, 1.92 M Glycine, and 1% SDS in distilled H2O) (make 500 mL)
- Molecular weight markers (Bio-Rad Precision Plus Dual Color; load 5-7.5 µL in a lane; store aliquot at -20∘C)
- Time point samples (spun down cell pellet from 250 mL of each expression)
- Stain /Destain solutions: will be provided for class at a station in the fume hood.
- Coomassie Brilliant Blue Stain G-250
- Methanol 40%
- Acetic Acid 10%