Western Blot protocol 

Making the gel
1. Use alcohol and Kimwipes to wipe the glass, and then set up the rest of the apparatus
2. For small proteins, use a higher percentage gel
2. Add all ingredients except ammonium persulfate; before adding ammonium persulfate, if the weather is cold in the room, you may wash the solution with hot/warm tap water to help the polymerization
3. Invert tube gently after adding all components
4. Add it up to the correct mark
5. Add t-butanol
6. Remove t-butanol, wash with water, and wipe with filter paper; put in comb
7. Make upper gel and gently shake 2x
8. Add upper gel
9. Remember to keep tubes to check if they’ve polymerised (cap the tube after pouring and see if the material in the tube has polymerised)
10. Label the lanes of the upper gel with a marker if required

Preparing and Loading Samples
1. Set up the gel in the tank and make sure the buffer isn’t leaking (if it is, it means that the glass plates are not slightly protruding up from the screws on both ends)
2. Pour in some running buffer to prevent drying of the gel
3. For each sample, use 1/3 the volume of 4x loading dye (e.g. if have 30uL of sample, add 10uL of 4x loading dye)
4. If running samples in reduced form, add 5% beta-mercaptoethanol in the fumehood
5. once the loading dye has been added, do no place on ice, or the SDS will precipitate out

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