Experiment #8 for CHM4304L -Protein Electrophoresis

Objectives

Protein Electrophoresis – To become familiar with SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins and to determine the molecular weight of T5 exonuclease.

Materials:

1. 30% Acrylamide/bis (29:1) solution;
2. 4 x Resolving gel buffer: 1.5 M Tris-HCl (pH 8.8), 0.4% SDS;
3. 4 x Stacking gel buffer: 0.5 M Tris-HCl (pH 6.8), 0.4% SDS;
4. 1 x Electrophoresis buffer: 25 mM Tris-HCl (pH 8.3), 250 mM glycine, 0.1% SDS.
5. 3 x protein loading buffer: 0.19 M Tris-HCl (pH 8.0), 3% glycerol, 6% SDS, 300 mM DTT, and 0.25% bromophenol blue;
6. 10% Ammonium persulfate (APS);
7. TEMED;
8. Gel staining buffer: 1 g of Coomassie Brilliant Blue dissolved in 400 ml of solution containing 45% methanol, 45% H₂O, and 10% acetic acid;
9. Destaining buffer: 25 % methanol, 10% acetic acid, and 65% H₂O;
10. Gel Drying Buffer: 40% ethanol, 4% glycerol, and 56% H₂O.

Procedure:

1. Load your samples:

Select the three samples from lab#6 that contained protein.  For all samples, mix 15 microliters of your sample with 4 microliters of 5 x sample loading buffer and 1 microliter 20 x reducing agent, and heat the samples at over 90 ⁰C for 5 min, and load 20 ml of your samples to each well of the gel. Also add the protein standards in two lanes on each gel.

2. Run the gels:

Apply power at 100-150 V until the dye has reached the top of the resolving gel, and then increase the power into 200 V. When the dye reaches the bottom of the resolving gel, turn off the power supplier. Disassemble the gel apparatus, and stain the gel for about one hour at Gel staining buffer. Destain your gel at the destaining buffer until the protein bands are clearly seen in the gel, and dry the gel if it is possible.

3. Prepare 40 ml of 15% resolving gel solution as following:

9.68 ml of H₂O

20 ml of 30% Acrylamide/bis solution

10 ml of 4 x resolving gel buffer

300 ml of 10% APS

20 ml of TEMED

Prepare 50 mL of 4X resolving gel buffer.  Assemble the gel caster (see http://www.proteomicsnijmegen.nl/FTMS_pages/Documents/protean3.pdf), and add the 15% gel mix to gel caster. Overlay the solution gently with water or 1-butanol saturated with H₂O, and allow to be polymerized. You should see the gel-H₂O interface disappear and then reappear when the gel is polymerized

4. Prepare 20 ml of 5% stacking gel solution as following:

11.5 ml of H₂O

3.3 ml of 30% Acrylamide/bis solution

5 ml of 4 x stacking gel buffer

200 ml of 10% APS

10 ml of TEMED

Prepare 50 mL of 4X stacking gel buffer.  Add the gel mix to the gel caster, and insert the comb to the gel solution; you should wait until gel is fully polymerized. Once the gel is polymerized, the comb can be removed gently. Then assemble the gel into the electrophoresis apparatus.

5. Determination of molecular weight of T5 exonuclease:

Measure the migration distance of your purified protein from the top of the resolving gel. Plot log value of molecular weight versus the migration distance of the standard proteins, and then determine the molecular weight of T5 exonuclease: