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From 2012.igem.org

Contents 1 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 1.1 Pouring the polyacrylamide gel 1.2 Preparing the sample 1.3 Running the gel 2 Liquid chromatography–mass spectrometry 2.1 Sample preparation 2.1.1 Substrate preperation 2.1.2 Degradation 3 Activity measurements

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

This analytical method can be used for separation and identification of proteins according to their electrophoretic mobility. The mobility is a function of length of the molecular weight. Proteins that have identical charge per unit mass due to binding of SDS results in an equal electrophoretic mobility.

Pouring the polyacrylamide gel

• Make a master mix for the stacking and separating gel without adding ammonium persulfate and TEMED.
• Aliquote 6,5 mL for each separating and 2,5 mL for each stacking gel.
• Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form. Leave about 2 centimeters below the bottom of the comb for the stacking gel.
• Layer isopropanol on top of the gel.
• Leave the separating gel at room temperature for >60 minutes to polymerize.
• Remove isopropanol and wait until the surface is dry.
• Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form.
• Insert comb without getting bubbles stuck underneath
• Leave the gel at room temperature for >60 minutes to polymerize.

• For storage
  • Remove sealing and store the gel wrapped in moistened paper towel at 4°C.

Preparing the sample

• Mix your protein mixture 4:1 with Laemmli-buffer (15 µL protein solution + 5 µL Laemmli-buffer)
• Heat for 5 minutes at 95 °C.

Running the gel

• Remove sealing, put the polymerized gel into gel box and pour SDS running buffer into the negative and positive electrode chamber.
• Remove comp without destroying the gel pocket.
• Pipet the sample into the gel pockets, adjusting the volume according to the amount of protein in your sample. Make sure to include a lane with molecular weight standards (5µl PageRuler Prestained Protein Ladder™ (Fa. Fermentas)) to determinate the molecular weight of your sample.
• Connect the power lead and run the stacking gel with 10 mA until the blue dye front enters the separating gel.
• Raise amperage up to 20 mA for running the separating gel.
• When the distance of the lowest molecular weight standard lane to the gel end is down to 0.5 cm stop the electrophoresis by turning off the power supply.

Liquid chromatography–mass spectrometry

This method can be used to seperate analyts. The seperated sample can then be analyzed with mass-spectrometry. We used this technique to identify degradation products after the Laccase treatment to our Substrates.

Sample preparation

Substrate preperation

Our Substrates are soluble in Methanol. We set our standarts into a concentration of 1 mg/ml. The detection limit for the LC-MS showed that a concentration of XXXX for the Substrates Esteron and Estradiol is the highest concentration of detection. Same limit of detection was used for Ethinestradiol and Anthracen for degradation. We only used thoose four Substrates. For all LC-MS preperations we used the T. versicolor Laccases.

Degradation

For the degradation of our Substrates we used following standard reaction:

Material	Volume
Robinson Buffer	400µL
Substrates	50µL
Laccase	50µL

The activation of the T. versicolor Laccase was set into 0,1U in 50µL.

Activity measurements

For the measurements [http://www.sigmaaldrich.com/catalog/product/sigma/m0312?lang=de&region=DE 96-well flat bottom microplates] were used. Each well contained a total sample volume of 200 µL respectively. The sample setup was pipetted as follows:

component	concentration
buffer:	100 mM
Laccase	0,1 U
[http://www.sigmaaldrich.com/catalog/product/sigma/a1888?lang=de&region=DE ABTS]	0,1 mM
H2O	ad 200 µL

The [http://www.sigmaaldrich.com/catalog/product/sigma/53739?lang=de&region=DE Laccase of T. versicolor] we bought for standardization was diluted in water so that 140 µL would contain 0,1 U meaning 72 x 10^-5 g of the enzyme. Please check the labjournal if Sodium-Acetate or Briton Robinson Buffer was used, respectively. The ABTS and laccase concentration optimum for standardization (so that the reaction was traceable nicely) was determinated during several experiments. Check the labjournal for further information.

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