Haynes:PAGE

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SDS-PAGE

by Karmella Haynes, 2015

Principle: Proteins are denatured and given a negative charge with a detergent (SDS), loaded to the top of a vertical gel, then separated by protein fragment size by applying an electric charge

MATERIALS

• Protein sample buffer (e.g. NuPAGE® LDS Sample Buffer (4X) )
• DL-Dithiothreitol (DTT) redox agent (Sigma D0632-1G)
• SDS Running buffer - 20x NuPAGE® MOPS SDS Running Buffer (Life Technologies NP0001)
• Antioxidant - NuPAGE® Antioxidant (Life Technologies NP0005)
• Polyacrylamide gel (use the appropriate gel for your application)
  • Bis-Tris - small to mid-size proteins
  • Tris-Acetate - large proteins

EQUIPMENT

• Vertical gel electrphoresis chamber (e.g.

PROCEDURE

1. Label enough 1.5 mL eppendorf tubes for one blank (1) , five standard samples (2-6), and all of your unknown samples (7-...n).
2. Add 500 μL Bradford Reagent to each tube. You will add protein to these later, and ignore the negligible change caused by the additional volume.
3. Dilute the stock BSA in a new tube to make 50 μL of 1 μg/μL BSA. Example: if the stock BSA is 10 mg/mL, add 5 μL of BSA to 45 μL dH₂O in a fresh tube.
4. Add a BSA standard protein solution to tubes 1-6. See Table 1.
5. Add 5.0 μL of unknown to each remaining tube. Keep track of your samples with good labeling!
6. Close all caps and invert the tubes to thoroughly mix the samples.
7. Transfer 200 μL of the blank (tube one) into the first well in a clear 96-well flat-bottom plate.
8. Do the same for the other samples, using new wells.
9. Use a plate reader to record absorbance at 590 nm (OD 590). If using the BioTek Synergy H1 Software, set up a new protocol and under Procedure > Action > Read use the following settings
   1. Detection Method = Absorbance
   2. Read Type = Endpoint
   3. Wavelength (1) = 590 nm (type-in the value manually)

Table 1. Standard sample set-up

Reagent	Tube 1 (0 μg BSA)	Tube 2 (1 μg BSA)	Tube 3 (2 μg BSA)	Tube 4 (4 μg BSA)	Tube 5 8 μg BSA)	Tube 5 (16 μg BSA)
Bradford Reagent	500 μL	500 μL	500 μL	500 μL	500 μL	500 μL
BSA diluted to 1 μg/μL	0 μL	1.0 μL	2.0 μL	4.0 μL	8.0 μL	16.0 μL

What to do with your data: calculate unknown protein concentration(s)

1. Subtract the blank OD 590 value (Tube 1) from all other values.
2. Plot a standard curve (using Excel) with BSA concentration (x-axis) vs. Absorbance at 590 nm (y-axis). See this example.
3. Add a line of best fit (not a curve) and display the equation.
4. Solve the equation for x. Substitute y with the background-subtracted OD 590 for the unknowns. The x value will be the protein concentration of the unknown as μg/5.0 μL (because you used 5 μL to set up the assay sample).
5. Convert the unknowns to μg/μL: (x μg/5.0 μL) / 5 = x μg/μL